X-chromosome inactivation in monkey embryos and pluripotent stem cells
Masahito Tachibanaa, Hong Maa, Michelle L. Sparmana, Hyo-Sang Leea, Cathy M. Ramseya,
Joy S. Woodwarda, Hathaitip Sritanaudomchaia,1, Keith R. Mastersona,2, Erin E. Wolffa,
Yibing Jiaa, Shoukhrat M. Mitalipova,b,c,d,n
aOregon National Primate Research Center, Oregon Health & Science University, 505 N.W. 185th Avenue, Beaverton, OR 97006, USA
bOregon Stem Cell Center, Oregon Health & Science University, 505 N.W. 185th Avenue, Beaverton, OR 97006, USA
cDepartment of Obstetrics and Gynecology, Oregon Health & Science University, 505 N.W. 185th Avenue, Beaverton, OR 97006, USA
dDepartment of Molecular & Medical Genetics, Oregon Health & Science University, 505 N.W. 185th Avenue, Beaverton, OR 97006, USA
a r t i c l e i n f o
Received 15 May 2012
Received in revised form
26 July 2012
Accepted 14 August 2012
Available online 23 August 2012
Embryonic stem cells
Inner cell mass
a b s t r a c t
Inactivation of one X chromosome in female mammals (XX) compensates for the reduced dosage of
X-linked gene expression in males (XY). However, the inner cell mass (ICM) of mouse preimplantation
blastocysts and their in vitro counterparts, pluripotent embryonic stem cells (ESCs), initially maintain
two active X chromosomes (XaXa). Random X chromosome inactivation (XCI) takes place in the ICM
lineage after implantation or upon differentiation of ESCs, resulting in mosaic tissues composed of two
cell types carrying either maternal or paternal active X chromosomes. While the status of XCI in human
embryos and ICMs remains unknown, majority of human female ESCs show non-random XCI.
We demonstrate here that rhesus monkey ESCs also display monoallelic expression and methylation
of X-linked genes in agreement with non-random XCI. However, XIST and other X-linked genes were
expressed from both chromosomes in isolated female monkey ICMs indicating that ex vivo pluripotent
cells retain XaXa. Intriguingly, the trophectoderm (TE) in preimplantation monkey blastocysts also
expressed X-linked genes from both alleles suggesting that, unlike the mouse, primate TE lineage does
not support imprinted paternal XCI. Our results provide insights into the species-specific nature of XCI
in the primate system and reveal fundamental epigenetic differences between in vitro and ex vivo
primate pluripotent cells.
& 2012 Elsevier Inc. All rights reserved.
X chromosome inactivation is believed to be an essential
mechanism regulating the dosage compensation of X-linked
genes in eutherian mammals so that females with two X chromo-
somes do not overexpress X-linked genes compared to males
(Lyon, 1961). XCI is initiated during early mouse preimplantation
embryo development, where the paternally inherited X chromo-
some is silenced in early cleaving embryos. However at the
blastocyst stage, paternal X chromosome is transiently reacti-
vated in the ICM, resulting in two active X chromosomes (XaXa).
However, paternally imprinted XCI is maintained in the mouse TE
lineage (Hajkova and Surani, 2004; Okamoto et al., 2004). Random
XCI takes place in the ICM lineage after implantation, at about the
time of gastrulation, through epigenetic silencing involving
XIST RNA coating of the inactive X in cis (Panning et al., 1997;
Penny et al., 1996). Thus, somatic tissues in females are mosaic
composed of two cell types expressing from one or the other
In contrast to this strict X gene dosage compensation mechan-
ism in the mouse, approximately 15% of X-linked genes in
humans escape XCI and are expressed biallelically in females
(Carrel and Willard, 2005). Why and how these escape genes are
transcribed from a largely inactivated X chromosome is not fully
understood. In addition, the existence of paternally imprinted XCI
in the TE lineage in humans remains controversial, where few
studies reported conflicting findings (Moreira de Mello et al.,
2010; Zeng and Yankowitz, 2003).
ESCs are in vitro pluripotent cell lines derived from the ICM of
preimplantation blastocysts in several species, including mice,
nonhuman primates, and humans (Evans and Kaufman, 1981;
Martin, 1981; Thomson et al., 1995; 1998). ESCs can be maintained
and propagated indefinitely in a pluripotent state providing an
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nCorresponding author at: Oregon National Primate Research Center, Oregon
Health & Science University, 505 N.W. 185th Avenue, Beaverton, OR 97006, USA.
Fax: þ1 503 690 5563.
E-mail address: firstname.lastname@example.org (S.M. Mitalipov).
1Current address: Department of Oral Biology, Faculty of Dentistry, Mahidol
University, Bangkok 10400, Thailand.
2Current address: University Fertility Consultants, Oregon Health & Science
University, 3303 S.W. Bond Avenue, Portland, OR 97239, USA.
Developmental Biology 371 (2012) 146–155
unlimited supply of undifferentiated cells for cell replacement
therapy. However, isolation of stable mouse female ESCs remains
problematic due to frequent loss of one of the two X chromosomes
(Zvetkova et al., 2005). In a few existing stable mouse XX ESCs, both
X chromosomes remain active and XCI is initiated upon differentia-
tion (Nichols and Smith, 2009).
In contrast to the mouse, isolation of male and female primate
ESCs is equally efficient and loss of one of the two X chromosomes
is relatively rare in human female ESCs. However, a majority of
human female ESC lines appear to have undergone XCI in an
undifferentiated state (Shen et al., 2008; Silva et al., 2008).
Moreover, these human ESCs often exhibit monoallelic expression
of X-linked genes, suggesting either imprinted XCI, as seen in the
mouse TE lineage (Shen et al., 2008), or random XCI followed by
the clonal selection of the one or another populations during ESC
isolation and culture.
It remains unclear whether such fundamental differences
between mouse and primate ESCs reflect species-specific differ-
ences in the tissue of origin. For example, XCI in human ESCs
could simply reflect the pre-existing status in the parental ICMs.
Alternatively, XCI may indicate epigenetic instability during
isolation and long-term culture of human ESCs. Our recent study
demonstrated that monkey ESCs are unable to contribute to
chimeras upon injection into host blastocysts (Tachibana et al.,
2012). However, transplanted ICMs formed viable fetuses while
sharing the TE compartment with host blastocysts. These results
necessitate further investigations into genetic and epigenetic
mechanisms responsible for such drastic differences in develop-
mental potential of primate ICMs vs. ESCs. Currently, few studies
are available on X inactivation status and timing in human
embryos (Okamoto et al., 2011; van den Berg et al., 2009). This
is in large part, due to restrictions on human embryo research and
the lack of relevant genetic markers that would allow discrimina-
tion of two X chromosomes.
To address this gap in the knowledge, we carried out a
comprehensive analysis of XCI on a clinically relevant nonhuman
primate model. We investigated allele specific expression and
methylation of several X-linked genes in female rhesus macaque
(Macaca Mulatta) blastocysts, focusing particularly on the ICM
and TE. We also extended our studies to rhesus monkey ESCs
derived from fertilized embryos or experimental pluripotent stem
cells derived by reprogramming of somatic cells using somatic
cell nuclear transfer (SCNT) or iPS (induced pluripotent stem) cell
Materials and methods
All animal procedures were approved by the Institutional
Animal Care and Use Committee (AICUC) at the ONPRC/OHSU.
Production of monkey embryos, ICM and TE isolation and gender
Rhesus macaque embryos were generated by intracytoplasmic
sperm injection (ICSI) and cultured to the blastocyst stage as
described previously (Wolf et al., 2004). In vivo developmental
competence of these embryos was previously demonstrated by
birth of healthy rhesus offspring (Tachibana et al., 2009; 2012;
Wolf et al., 2004). Intact ICMs from various stage blastocysts were
isolated by immunosurgery (Mitalipov et al., 2006). In brief, zona
pellucidae were digested by short (10 s) treatment with 0.5%
protease. Blastocysts were then incubated in anti monkey whole
serum (Sigma) for 30 min at 37 1C, washed three times with
culture medium and transferred into guinea pig complement
(Sigma) for 30 min. Blastocysts were then gently pipetted with a
small-bore pipette to disperse lysed TE cells and isolate intact
ICMs. For the TE isolation, a zona-free blastocyst was held with
a micropipette near the ICM. Next, a sequential laser pulse
(Staccato laser www.hamiltonthorne.com) was fired across the
boundary between the ICM and TE while the TE part was pulled
away with a second pipette until complete separation. Unutilized
ICM or TE cells were used for gender determination using PCR
approach. Cells were collected into 0.2 ml PCR tubes containing
4 ul of PicoPuresDNA (Arcturus Bioscience) extraction buffer,
and X- and Y-linked zinc finger protein genes (ZFX and ZFY) were
amplified as previously described (Mitalipov et al., 2007). Female
samples produced 1149 bp fragment while male samples con-
tained an additional 771 bp fragment.
Derivation, culture and characterization of monkey iPS cells
Primary cultures of fibroblasts were established from rhesus
macaque skin biopsies. Fibroblasts in the log growth phase were
transduced with retroviral vectors carrying 4 transcription factors as
previously described (Takahashi et al., 2007; Wu et al., 2009).
Briefly, plasmids (pMXs–hOCT4, pMXs–hSOX2, pMXs–hKLF4 and
pMXs–hC–MYC, Cell Biolabs, Inc. San Diego, CA) were packaged into
retroviral particles by transfection into Platinum-A Retroviral Packa-
ging Cells using FugenesHD Transfection Reagent (Roche, Indiana-
polis, IN). Transduction of fibroblasts was performed three times at
24 h intervals, followed by seeding of cells onto feeder layers of
mitotically inactivated mouse embryonic fibroblasts (mEFs) in ESC
culture medium consisting of DMEM/F12 medium with high glu-
cose, without sodium pyruvate and supplemented with 1% nones-
sential amino acids, 2 mM l-glutamine, 0.1 mM b-mercaptoethanol
and 15% FBS (Mitalipov et al., 2006). The transduced cells were
maintained at 37 1C in 3% CO2, 5% O2and balance N2for up to
4 weeks or until colonies of cells with a morphology similar to ESCs
appeared. Each colony was then individually isolated and manually
propagated using standard ESC culture techniques as previously
described (Byrne et al., 2007; Mitalipov et al., 2006; Sparman et al.,
Expression of ESC markers in iPS cells was detected by
immunocytochemistry as previously described (Mitalipov et al.,
2006; Sparman et al., 2009). Primary antibodies for OCT4, SSEA-4,
TRA-1-60 and TRA-1-81 were from Santa Cruz Biotechnology Inc.
and NANOG was from R-D Systems.
Comparative microarray analysis of mRNA profiles in iPS
cells and their IVF or SCNT controls was carried out using
the Affymetrix Rhesus Macaque Genome array as previously
described (Sritanaudomchai et al., 2010). RNA samples were
converted to labeled cRNA and hybridized to GeneChip Rhesus
Macaque Genome Arrays (Affymetrix, Inc.). Gene-Chip operating
system version 1.4 software (Affymetrix) was used to process
images and generated probe level measurements. Microarray
data, including CEL and CHP files, can be accessed at the Gene
Expression Omnibus (GEO: GSE36252), http://www.ncbi.nlm.nih.
Processed image files were normalized across arrays using the
robust multichip average algorithm (Irizarry et al., 2003) and log
transformed (base 2) to perform direct comparisons of probe set
values between samples. GeneSifter (VizX Labs, Seattle, WA)
microarray expression analysis software was used to identify
differentially expressed transcripts. For a given comparison, IVF-
derived ESCs were selected as the baseline reference, and tran-
scripts that exhibited various fold change relative to the baseline
were considered differentially expressed. To facilitate in-depth
comparisons, processed image files were normalized with the
robust multichip average algorithm and log transformed (base 2)
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