Download Derivation of naïve human embryonic stem cells

Derivation of naïve human embryonic stem cells
Carol B. Warea,b,1, Angelique M. Nelsona,b, Brigham Mechamc, Jennifer Hessona,b, Wenyu Zhoua,d, Erica C. Jonlina,e,
Antonio J. Jimenez-Caliania,f, Xinxian Dengg, Christopher Cavanaugha,b, Savannah Cooka,b, Paul J. Tesarh,
Jeffrey Okadaa,e, Lilyana Margarethaa,e, Henrik Sperbera,i, Michael Choia,i, C. Anthony Blaua,e, Piper M. Treutingb,
R. David Hawkinsa,j, Vincenzo Cirullia,f, and Hannele Ruohola-Bakera,d,i
a
Institute for Stem Cell and Regenerative Medicine, Departments of bComparative Medicine, dBiology, and iBiochemistry, and Divisions of eHematology,
Metabolism, Endocrinology and Nutrition, and jMedical Genetics, Department of Medicine, University of Washington, Seattle, WA 98195; cSage Bionetworks,
Seattle, WA 98109; gDepartment of Pathology, University of Washington, Seattle, WA 98195; and hDepartment of Genetics, Case Western Reserve University,
Cleveland, OH 44106
f
Edited* by Stanley M. Gartler, University of Washington, Seattle, WA, and approved February 12, 2014 (received for review October 24, 2013)
The naïve pluripotent state has been shown in mice to lead to
broad and more robust developmental potential relative to primed
mouse epiblast cells. The human naïve ES cell state has eluded
derivation without the use of transgenes, and forced expression
of OCT4, KLF4, and KLF2 allows maintenance of human cells in
a naïve state [Hanna J, et al. (2010) Proc Natl Acad Sci USA 107
(20):9222–9227]. We describe two routes to generate nontransgenic naïve human ES cells (hESCs). The first is by reverse toggling
of preexisting primed hESC lines by preculture in the histone
deacetylase inhibitors butyrate and suberoylanilide hydroxamic
acid, followed by culture in MEK/ERK and GSK3 inhibitors (2i) with
FGF2. The second route is by direct derivation from a human embryo in 2i with FGF2. We show that human naïve cells meet mouse
criteria for the naïve state by growth characteristics, antibody labeling profile, gene expression, X-inactivation profile, mitochondrial morphology, microRNA profile and development in the
context of teratomas. hESCs can exist in a naïve state without
the need for transgenes. Direct derivation is an elusive, but attainable, process, leading to cells at the earliest stage of in vitro pluripotency described for humans. Reverse toggling of primed cells
to naïve is efficient and reproducible.
I
t has become clear with the derivation of mouse epiblast stem
cells (mEpiSCs) that pluripotency encompasses more than one
stage of development (1, 2). The earlier “naïve” stage represents
the preimplantation inner cell mass, typified by mouse ES cells
(mESCs), and the “primed,” the postimplantation epiblast, typified by mEpiSCs and human ES cells (hESCs). The challenge in
naïve cell maintenance has been protecting cells from differentiation stimuli. This has been achieved in mESCs through exposure to leukemia inhibitory factor (LIF), whereas addition of
extracellular signal-regulated kinase (MEK) and glycogen synthase kinase 3 (GSK3) inhibitors (2i) in defined medium allows
the cells to attain a homogeneous ground state (3). Defining
characteristics of the naïve/ground vs. primed states are shown in
Fig. S1A. In humans, the naïve stage has been difficult to capture
as a stable in vitro state.
There are practical advantages that come with a human naïve
state. Among them is ease of trypsin passage and developmental
capacity. Whole animals can be generated from good naïve mESCs
through tetraploid complementation (4), and mEpiSCs cannot
contribute to chimerism. Being more comparable to mESCs, naïve
hESCs will likely allow increased developmental potential and
a more accurate correlation to mESC data.
It has been reported that human induced pluripotent cells
(h-iPSCs) can be maintained in the naïve state if the pluripotency-inducing transgenes are not silenced (5). Only recently
have hESCs been maintained in a naïve state without transgenes
(6). Our primary aim was to generate naïve hESCs not dependent upon transgenes for stable culture. We toggled existing
human ESC and mouse mEpiSC lines back from the primed
state to grow under the influence of 2i without the need for
Activin A. This helped us to define appropriate culture conditions for human naïve cells and allowed the de novo derivation
4484–4489 | PNAS | March 25, 2014 | vol. 111 | no. 12
of a naïve hESC line, Elf1. We report on the naïve state of human ESCs capable of unlimited culture in 2i.
Results
Primed Mouse and Human Pluripotent Cells Acclimate to Naïve 2i
Culture. In an earlier report, we described toggling mouse and
human ESCs to an earlier state using a histone deacetylase inhibitor (HDACi) (7). Thus, we used this approach as a stepping
stone to reverse primed cells to a naïve state. mEpiSCs and
hESCs were converted to 2i culture following one to three passages in a mixture of HDACi consisting of sodium butyrate and
suberoylanilide hydroxamic acid (SAHA, vorinostat) (B/S); nomenclature is explained in Methods). Without preexposure to
B/S, the majority of the primed stage mouse and human cells
differentiated following exposure to 2i (Fig. 1 A and B and Fig.
S1 B–G), as expected (3). Once cells were passaged through B/S,
both mouse and human responded to 2i through formation of
tight colony edges reflective of mESCs (Fig. 1B and Fig. S1B).
These human cultures remained dependent on basic fibroblast
growth factor (FGF2; F). All stages of pluripotency reflected
under these culture conditions express POU class 5 homeobox
1 (OCT4), Nanog homeobox (NANOG), and the cell surface
proteins TRA-1-60 and TRA-1-81. Unlike naïve mESCs, both
naïve and primed hESCs are stage-specific embryonic antigen-4
(SSEA4)-positive while remaining SSEA1-negative (Fig. S2A).
Primed H1, intermediate H1 (in B/S), and naïve H1 (in 2iF)
Significance
We report on generation of nontransgenic, naïve human pluripotent cells that represent the developmentally earliest state
described for human established cells. Existing human ES cell
lines in the later primed state can be toggled in reverse to naïve
by exposure to histone deacetylase inhibitors prior to naïve
culture. A new line was established directly from an eight-cell
embryo under naïve culture conditions. We describe the naïve
state in humans and show that naïve human ES cells have
expanded endoderm developmental capacity.
Author contributions: C.B.W., W.Z., C.A.B., R.D.H., and H.R.-B. designed research; C.B.W.,
A.M.N., J.H., W.Z., A.J.J.-C., X.D., C.C., S.C., J.O., H.S., M.C., R.D.H., and V.C. performed
research; E.C.J. and P.J.T. contributed new reagents/analytic tools; C.B.W. and A.M.N.
obtained consent for embryo donation; E.C.J. obtained human subjects’ approval/consent; C.B.W., B.M., W.Z., J.O., L.M., H.S., P.M.T., R.D.H., and V.C. analyzed data; and
C.B.W. wrote the paper.
The authors declare no conflict of interest.
*This Direct Submission article had a prearranged editor.
Freely available online through the PNAS open access option.
Data deposition: The Agilent array data reported in this paper has been deposited in the
Synapse Commons Repository database, https://synapse.sagebase.org/ [accession nos.
syn1447097 (H1-2iF vs. H1), syn1447098 (Elf1-3iL vs. Elf1-AF), and syn1447101 (mESC vs.
mEpiSC vs. mEpiSC-2iL)].
1
To whom correspondence should be addressed. E-mail: [email protected].
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1319738111/-/DCSupplemental.
www.pnas.org/cgi/doi/10.1073/pnas.1319738111
would represent altered states as determined by expression. The
difference between right and left quadrants would be more
profound than the difference between top and bottom. Although
the toggled naïve mESCs [mEpiSC-(B/S)2iL] have the morphological appearance of mESCs (Fig. S1B), these cells are more
similar to delayed-implantation icm (Fig. 2B). Thus, there is
likely an advantage to direct naïve cell derivation. Therefore, we
generated a new hESC line from a donated embryo under the
naïve culture conditions determined by H1-2iF cells.
Generation of a New Naïve hESC Line. One eight-cell embryo
formed what seemed to be a naïve hESC line, with mounded
colony morphology having bright edges (Fig. S1I). The efficiency
cultures grown on Matrigel were karyotyped by G-banding at
high passage number (passage 93). H1 grown in B/S (H1p93B/
S45) and grown in B/S followed by 2iF [H1p93(B/S17)2iF27]
displayed a normal 46XY karyotype (5/5 spreads analyzed each).
The companion cells [H1p93(M12)T19], grown in the primed
state throughout, gained genetic material with all cells carrying
trisomy 12 and trisomy 17 (5/5 spreads) and some of those carrying an additional marker chromosome of unknown origin (3/5).
To explore the biological effect of LIF, naïve H1s were exposed
to human LIF or FGF2. Cells that were supported on feeders
with LIF exhibited a naïve mESC-like morphology by mounding,
whereas colonies were flatter in FGF2 (Fig. S1 C–F). Despite the
naïve mESC morphology following one passage, H1-2iL began
showing signs of differentiation by the third passage and were lost
to differentiation by the third to fifth passage (Fig. S1G).
Along with five hESC lines (H1, H7, H9, HUES1, and HUES2),
two mEpiSC lines (mEpiSC5 and mEpiSC7, 2) and two human
induced pluripotent lines were converted to 2i culture. In addition,
one nonhuman primate line (MF-1; Macaca fascicularis; a gift of
Eliza Curnow, Washington National Primate Research Center,
Seattle) was converted to 2i culture (Fig. S1H). Unlike hESCs, the
nonhuman primate line could survive over several passages in LIF
with no FGF2 and did not require HDACi to reverse toggle, indicating a species difference between humans and M. fascicularis.
Gene Expression of H1 Cultured As Naïve Indicates an Earlier Pattern.
We analyzed the differences between the stages of human pluripotency, primed and toggled naïve through Agilent whole human genome array comparisons. Hypoxia inducible factor 2α
(HIF2α) (EPAS1) seems to be diagnostic of the 2iF stage in that
a significant number of HIF2α targets show a unique expression
pattern in naïve H1 cultures relative to primed (Fig. 2A; hypergeometric distribution, P < 0.01).
To determine whether cells that were toggled through B/S to
2i could be defined as “naïve” we used principal component
analysis. Microarray expression data derived from the mEpiSCs,
mEpiSCs converted to 2iL, and mESCs were compared with
a mouse developmental yardstick (8) (GSE8881), in which inner
cell mass (icm) was collected from mouse embryos at preimplantation (E3.7; thought to be equivalent to naïve mESC),
delayed implantation icm of diapause (E7.5, i.e., 3-d delay), and
postimplantation epiblast (E6.5). Clustering within a quadrant
Ware et al.
DEVELOPMENTAL
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Fig. 1. Effect of 2i culture on mouse and human pluripotent cells. (A) Alkaline phosphatase stain of mouse pluripotent colonies. Left two plates: 2i
added to mEpiSC colonies causes differentiation and loss of the alkaline
phosphatase positive cells. Right four plates: mEpiSCs grown in butyrate plus
SAHA for a minimum of 1 passage before addition of 2i allows pluripotent
colonies to flourish. (B) hESC (H1) toggled backward on Matrigel to 2i through
butyrate or forward to differentiation when not exposed to B/S before 2i
culture, indicating that 2i must follow B/S exposure to prevent differentiation.
(Scale bars, 100 μM.)
Fig. 2. Genomic analysis of naïve hESCs. (A) RNA expression heat map of
HIF2α (EPAS1) target genes in H1-2iF cells relative to parent H1 [H1p58
(B/ST2)2iF14 vs. H1p58 in TeSR2; run in quadruplicate]. (B) Principal component analysis comparison of mouse whole genome Agilent array data
comparing Hunter et al. (8) embryo data (Left) to mESC equivalents: R1p22
(mESC-2iL, naïve), mEpiSC7p24AF (mEpiSC-AF, primed), and mEpiSC7p55
(AF7,B/S1)2iL20 (mEpiSC-2iL, toggled to naïve) (Right). Elf1 naïve (3iL, green
squares) and primed (AF, blue squares) expression data are compared with
the in vivo mouse embryo data in the plot on the left. (C) Comparison of inhouse Elf1 expression array data as the standard against which to measure
in-house (UW) data (H1-2iF × four repetitions; primed: H1 × four repetitions)
and that generated by Hanna et al. (5). Naïve: C1.1, C1.2, WIBR3.1, WIBR3.3,
WIBR3.5; primed: first grouping-BG01, BG01-mTeSR; BG01-NANOGtgk; second grouping-WIBR1, WIBR2, WIBR3rep1, WIBR3rep2, and WIBR3–5%O2.
Note that the lines tested on left side that are compared with naïve Elf1 are
grouped identically on the Elf1 primed side of the graph. Presumed naïve
cell lines are represented by dark blue dots and presumed primed are orange. (D) DNase I hypersensitivity analysis of the POU5F1 enhancer regions
for Elf1 (lower line black) and H1 (line above in blue). The first exon of
POU5F1 is shown above the H1 data along with a 2-kb size bar to map the
proximal enhancer (PE) and distal enhancer (DE). (E) ChIP-seq H3K27me3
comparison of primed hESCs [orange line, data taken from Gafni et al. (6)] to
naïve Elf1-2iL (blue line) using the genes in C that intersect with Gene Ontogeny “developmental genes” (n = 648).
PNAS | March 25, 2014 | vol. 111 | no. 12 | 4485
of establishing a human pluripotent line in 2i was quite low at one
line from 128 donated embryos, primarily frozen as blastocysts.
Elf1 is positive for OCT4, NANOG, TRA-1-60, TRA-1-81, and
SSEA4 while remaining negative for SSEA1 (Fig. S2A), consistent
with recent reports (5, 6). The line was karyotyped at passage 9
and found to be a normal female (Fig. S2B).
Although Elf1 was started in 2iF, we attempted to convert it to
3i (includes an FGF receptor inhibitor) + human LIF, to determine whether Elf1 could grow independently of FGF2. Elf13iL resulted in a more mounded morphology characteristic of
mESC (Fig. S1I). Unlike H1-2iF, Elf1-3iL could be stabilized
without FGF2. Elf1 could be maintained as naïve indefinitely in
3iL and 2iL and as such has been grown stably for more than 60
passages. However, when FGF is not present there is an increased background of differentiated cells. There was negligible
culture drift when maintained in 2iF.
Two questions concerning culture arose. What is the operative
signaling mechanism of FGF2 in the presence of an MEK inhibitor? Mammalian target of rapamycin (mTOR) survival signaling is a possible alternative pathway used by FGF2. Both
naïve and primed Elf1 were sensitive to mTOR inhibition
through exposure to rapamycin (Fig. S3A). The second question
concerns the role of the Janus kinase/signal transducers and
activators of transcription (JAK/STAT) pathway in naïve Elf1
cells. A STAT3 inhibitor (NSC74859) was added to the 2iL
medium, which caused naïve cells to slow growth, whereas Elf1s
induced to the primed stage through Activin and FGF (AF) were
not obviously affected (Fig. S3B).
Elf1 Gene Expression Indicates the Line Is Naïve. Using the Sage
resources, Agilent expression data were compared with archived
array data. A set of 628 naïve Elf1 specific enriched (fourfold or
greater) genes and of 639 primed Elf1 specific enriched genes
were used as a yardstick against which to compare our data with
that in the literature. Naïve Elf1 expression, but not primed Elf1,
was found to correlate with previously reported naïve h-iPSC
expression using persistent expression of transgenes (5) (Fig.
2C). All of the primed hESC lines showed an expression signature that was clearly different from the naïve signature. Furthermore, cell lines treated as primed showed the most variability
in expression pattern. This could partially explain some of the
biological variability seen among primed hESCs.
The Elf1 array data were compared with the same previously
generated mouse embryo data used in Fig. 2B. The naïve Elf1
cells did fall into the same quadrant with mouse peri-implantation icm as seen in delayed implantation embryo analysis
whereas primed Elf1s reflected mEpiSCs (Fig. 2B). This is remarkable given the diversity of expression between mice and
humans and the differences in experimental process, indicating
that Elf1s are within the mouse naïve definition by expression.
Curiously, primed Elf1 cells could be switched back to naïve
conditions (2iL) without the need for a B/S intermediate step,
indicating a fundamental difference between hESCs derived
under naïve conditions, but cultured as primed, and hESCs
generated initially as primed cells.
MicroRNA (miR) pattern change is implicated during initiation of development. For this reason, we decided to look at select
miRs, miR-302b, miR-372, miR-518b, miR-520b, and miR520c.
These miRs are expressed in the primed state of human pluripotency (9, 10). miR-302b is thought to remain relatively stable
throughout human pluripotency and was used as a positive
control for the pluripotent state. miR-372 may increase in hESC
developmentally earlier than primed (10, 11). As part of a primate-specific chromosome 19 miR cluster (CM19C), miR-518b,
miR-520b, and mir-520c are known to be expressed in the
primed state and to decline upon differentiation (9). Thus, an
increase in these miR levels relative to miR 302b would indicate
that the cells are earlier in pluripotent development. miR-302b
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Fig. 3. Analysis of hESC stages of pluripotency. (A) MicroRNA analysis associated with pluripotency. (B) XIST labeling shows that Elf1-3iLs (Left) do
not display a XIST cloud by FISH, whereas Elf1s primed have two XIST signals
and cells differentiated for 10 d (Right) gain a single XIST signal (red dot)
within the nucleus. When the nucleus is highlighted using DAPI and the field
magnified XIST remains undetectable in naïve Elf1 (Lower Left), whereas the
XIST signal can be detected upon differentiation on one or both X chromosomes (red dots, white arrows, Lower Right two panels). (C) Bisulfite
sequencing of the XIST promoter using the 11L-11R primers shows that XIST
remains methylated throughout the naïve and primed stages. Using the
7L-7R primers, methylation seems to diminish in naïve relative to primed
cells, 10-d in vitro-differentiated cells and in the 98-d teratoma. Note that
the 1, 9, and 11 primer sets did not vary from data shown for set 11 upon
differentiation. Open circles indicate unmethylated and filled circles indicate
methylated CpGs. (D) Graphs representing cloning efficiency (percent) and
doubling times (hours) of Elf1 naive (green), Elf1 primed (yellow), H1 naïve (dark
blue), and H1 primed (light blue). (E) Electron microscopy of mitochondria. The
left panels show the mitochondrial shape difference between Elf1-3iL and Elf1AF. This is quantified in the graph on the right, where increased ratio indicates
a rounder mitochondrial population (± SEM). ***P < 0.001, **P < 0.01, and *P <
0.05 as determined by t test.
did remain reasonably stable in naïve and primed hESCs (Elf1,
H1, and H7), as expected. Expression of miRs 518b and 520b
and 520c increased in the naïve state relative to primed while
miR-372 expression increased in Elf1 relative to H1 and H7
(Fig. 3A).
To compare our ELF1 data to a recent report (6) on naïve
hESCs we looked at two parameters, POU5F1 (OCT4) enhancer
use and presence of H3K27me3. Naïve cells depend upon the
distal OCT4 enhancer, whereas primed cells use the proximal
enhancer (5, 6). We detected a similar phenomenon using
DNase I hypersensitivity analysis, which is useful for pinpointing
poised and active regulatory regions. Increased hypersensitivity
mapped to the distal enhancer in Elf1 and to the proximal enhancer in H1 (Fig. 2D). Recently, naïve hESCs were shown to
exhibit reduced H3K27me3 at developmental gene promoters
(6). We generated H3K27me3 ChIP-seq data in Elf1 naïve conditions and compared this to published H3K27me3 hESC-primed
levels (6) at 648 developmental genes used for comparison in Fig.
2C. We see an overall reduction of H3K27me3 signal in naïve cells
(P < 2.2 × 10−16), in agreement with previous findings.
Ware et al.
Elf1 Cells Have Two Active Xs with Appropriate X Inactivation on
Differentiation. Elf1 is a female line, which allowed us to ex-
plore X inactivation. FISH analysis for X-inactive specific transcript (XIST) indicated the absence of a signal in naïve Elf1. Two
XIST clouds arose in primed cells, and two clouds were present
in some of the cells upon random differentiation for 10 d in FBS,
but there were also cells with only one cloud (Fig. 3B). Identification of both Xs via XIST-FISH before X inactivation is
consistent with a previous observation on human embryos (12).
Thus, we looked at XIST promoter methylation as an indicator
of an inactive X, using four primer sets (13). Sets 1L-1R, 9F-9R,
and 11F-11R indicated similar extensive promoter methylation in
the naïve, primed, and differentiated states. The promoter region
detected by the 7L-7R set indicated demethylation of XIST in
naïve cells relative to primed and differentiated states. Absence
of X inactivation may, in part, be a feature of low oxygen culture,
as previously described (13).
Naïve hESCs Double More Rapidly and Clone More Efficiently Than
Primed hESCs. We measured both cloning efficiency and pop-
DEVELOPMENTAL
BIOLOGY
ulation doubling time in Elf1 naïve (2iL), Elf1 primed (AF), H1
naïve (2iF), and H1 primed (Fig. 3D). Elf1 naïve cloning efficiency was twice that of the other lines, and doubling time was
halved relative to H1. Elf1 primed and H1 naïve characteristics
are intermediate. The trend seen is similar to that reported
(5, 6), although absolute numbers are not identical, likely owing
to technical differences in the assays.
Mitochondrial Metabolism As a Functional Marker of Pluripotency
Stage. mESCs are bivalent and can convert between mitochon-
drial and glycolytic metabolism depending on culture conditions,
whereas mEpiSCs and hESCs are dependent primarily on aerobic glycolysis (14). Consistent with the mouse data, expression
of cytochrome c oxidase assembly protein and estrogen receptorrelated receptor B were significantly higher in the naïve Elf1
cultures relative to primed Elf1 (P < 1 × 10−4 and P < 0.01,
respectively; Agilent array data), signifying effective mitochondrial oxidative metabolism. Primed Elf1 increased RNA expression of HIF1α targets lactate dehydrogenase A, phosphoinositidedependent kinase-1 (PDK1), and phosphorylase, glycogen, liver
(PYGL) (P < 10−5, P < 0.01, and P < 0.01, respectively). HIF1α is
known to play an instrumental role in support of aerobic glycolysis.
The dependence of the primed state on glycolysis likely confers
a growth advantage that may be correlated to that seen in cancer
cells through the Warburg effect. PDK1, 2, and 3 are all up-regulated in primed Elf1 cells (P < 0.01, P < 0.01, and P < 10−5, respectively), indicating that some of the mechanism of primed cell
mitochondrial shutdown may be similar to that seen in the
Warburg effect (15, 16).
Mitochondrial morphology was assessed in naïve Elf1 and
primed Elf1 by electron microscopy. Reflective of the mouse
naïve vs. primed cells (14), naïve Elf1 mitochondria appeared
less mature, in that they were generally round with few cristae,
whereas mitochondria in primed cells were elongated with
somewhat more developed cristae (Fig. 3E).
Fig. 4. Teratomas generated from naïve, naïve toggled to primed, and
primed toggled to naïve cells reflect extensive endoderm developmental
capacity. (A) H&E-labeled sections of Elf1p17-2iL10 (naïve; 42 d) and
Elf1p15T8 (primed, 67 d) teratomas. (B) Endoderm-specific labeling of sections from the Elf1 teratomas shown in A. The upper two panels of both
tumors are sequential sections, the upper labeled to highlight liver development (red, albumin; green, α-fetoprotein; and blue, E-cadherin) and
the second set to highlight pancreatic development (red, PDX1; green, SOX9;
and blue, E-cadherin). The next three panels (descending) for both tumors
also represent different sequential sections with the first set representing
liver development, the second set pancreatic development, and the third set
liver development by alternative markers (labeled as above and red, CYP3A
and green, HNF4A). The lower Elf1p17-2iL10 naïve panel are in place to
reinforce the impression of the level of organization of endodermal development within these tumors (red, FOXA2; green, SOX9; and blue,
E-cadherin), and the bottom right panel (Elf1p15T8) is a negative control.
(C ) H&E sections of an H1 naïve, 44-d teratoma. The graph below the H&E
sections represents quantitation of the areas stained for either E-cadherin
(epithelial cells) or PDX1 (pancreatic progenitors) in primed (H1p44-AF9),
naïve [H1p49(B/S3)2iF10], and naïve reverted to primed [H1p49(B/S3, 2iF4)
AF5] H1 generated teratomas, indicating that total epithelial developmental
potential and the pancreatic subset are both enhanced in the naïve state
relative to primed. (D) Top three panels are H&E sections of an mESC teratoma (naïve, 13 d). The lower panels show immunofluorescent labeling of
sections from this mESC teratoma. (Scale bars in A and D, 100 μM; they define the scale for all H&E-stained sections.)
Teratoma Formation. Teratoma formation has conventionally
been used in pluripotent cell assessment to determine the
ability of a line to generate the three germ lineages. Representatives of the three germ lineages are present in teratomas
from toggled and de novo-derived naïve hESCs, proving their
pluripotency (Fig. 4). This assay has the potential to highlight
the lineage differentiation potential of any individual pluripotent line. It is in this regard that we pursued teratoma formation to determine the endoderm lineage contribution.
Tumors were harvested from animals injected with naïve Elf1
cells within 42–50 d and primed Elf1 by 67 d, when they were
first detectable by palpation. Studies by others with primed
hESC-generated teratomas have noted the disproportionate
amount of mesoderm in the tumor mass (amounting to between
two-thirds and three-fourths of the tumor) (17, 18). As shown in
Fig. 4B, teratomas derived from both naïve and primed Elf1s
contained significant compartments occupied by E-cadherin–
positive areas whose phenotype could be clearly ascribed to
definitive endoderm cell lineages owing to the proximity to the
developing liver. Specific examples are shown for the liver cell
lineage, marked by the expression of albumin, α-fetoprotein, and
cytochrome P450, family 3, subfamily A (CYP3A), whereas in
adjacent sections pancreatic cell lineage marked by the expression
of pancreatic and duodenal homeobox 1 (PDX1) and SOX9 in
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E-cadherin–positive cells seems associated with, yet separate from,
liver development. Immunoreactivity for hepatocyte nuclear factor
4, alpha (HNF4A), CYP3A, forkhead box A2 (FOXA2), and/or
sex determining region Y, box 9 (SOX9) also identifies regions
occupied by more immature common progenitors for liver and
pancreas lineages. This phenotype contrasts with that observed in
teratomas derived from primed H1 cells, in which clusters of immature endoderm were reduced relative to naïve H1. E-cadherin
immunoreactivity, indicating epithelial cells and PDX1 immunoreactivity, indicating pancreas progenitors, were both increased in
the naïve H1 (Fig. 4C, graph). Reversion of naïve cells to primed via
culture reduces both E-cadherin– and PDX1-positive areas. Lineage
marker data were combined for H1 and Elf1 naïve (n = 6) vs. primed
(n = 4) teratomas (Fig. S4). Endoderm was increased in the naïve cellgenerated tumors explored relative to the two primed cell-generated
tumors, whereas neural and mesenchymal lineages were reduced.
Because mESCs are the naïve gold standard, we assessed
mESC teratomas. These were harvested 13 d following injection.
mESC-generated tumors looked similar to hESC-generated
tumors. Endoderm was well developed (Fig. 4D), although
immature, relative to day-42 to day-67 naïve human teratomas.
Overall, the teratoma data indicate that robust endoderm development is attenuated when hESCs are derived under primed
conditions. Lineage potential seems to be protected throughout
in vitro pluripotent culture if hESCs are derived as naïve. Robust lineage potential is regained when H1s are toggled backward through HDACi to a naïve state.
Discussion
We show that naïve human pluripotent cells can be derived directly
from the preimplantation human embryo and that hESCs derived
beyond a point in the transition from naïve to primed maintain the
ability to become stably naïve in response to culture conditions.
This is supported by a recent report (6) that, in addition to 2iLF,
used increased MAP kinase pathway inhibition through p38 and
JNK inhibition along with transforming growth factor beta
(TGF-β) addition to allow reverse toggling and maintenance of
de novo-derived naïve hESCs. In our hands, reverse toggling of
primed hESCs is facilitated by HDACi exposure in humans,
whereas in mice naïve cells can arise directly from mEpiSC cultures
under naïve conditions (19). However, we see that preculture in
HDACi assists mEpiSCs in surviving initial exposure to 2i.
The ability to maintain reverse-toggled hESCs in a naïve state
seems to hinge around the continued presence of FGF. Human
response to FGF2 does not completely phenocopy the mouse
during the naïve-to-primed transition. Unlike in mice, human hypoblast formation does not require FGF2 (20), whereas FGF2
response in the presence of MEK/ERK inhibitor indicates that
FGF is likely to have a positive effect on self-renewal in both the
naïve and primed states, although perhaps through different signaling pathways. Active MEK signaling is important in the primed
state, whereas phosphoinositide 3-kinase/mTOR signaling or another FGF-induced cell signal suppresses differentiation in both
the naïve and primed states. In support of this, mouse primordial
germ cells require a 1-d exposure to FGF2 to toggle to pluripotency, and forskolin can substitute for FGF2 in this context (21).
Because human iPSCs can survive as naïve if cultured continuously
in forskolin (5), this suggests that forskolin can, at least partially,
substitute for continuous presence of FGF2 to stabilize hESC in
the naïve state. We have been unable to generate a new hESC line
directly in 2iL, and the one line generated in 2iF was thawed as an
eight-cell embryo. The presence of FGF2 and/or the stage of development at freeze/thaw may have been crucial to halting development in the naïve state. The extremely poor direct derivation
rate of naïve hESCs from embryos may be due to culture conditions marginal with regard to halting human icm development.
Increased inhibition of MAP kinase signaling, in addition to MEK
inhibition through the use of p38 and JNK inhibitors (6), may
4488 | www.pnas.org/cgi/doi/10.1073/pnas.1319738111
improve naïve hESC derivation rate. We note that the amount of
GSK3i used influenced culture stability such that cells selfrenewed in 0–2 μM GSK3i, whereas 5 μM drove differentiation.
This varies from mESCs, where 3 μM GSK3i is optimal (3), but is
similar to observations made with naïve rat ESCs, for which 1 μM
GSK3i is optimal (22, 23). Excess GSK3i may enable Wnt-induced
nuclear events that are counter to the naïve state.
A key advantage of naïve hESCs is the predicted ability to
better reflect development. This expectation has been borne out
in the teratoma assays. Generation of the endoderm lineage is
particularly robust from naïve cells. We interpret this to mean
that some tissue restriction for teratoma formation occurs when
hESCs are established in the primed state. A caveat to claiming
that teratoma developmental potential reflects fetal development
arises out of a recent paper (24) showing that mouse cells derived
as primed mEpiSCs can integrate correctly into fetal tissue if introduced into epiblast stage embryos. However, given the nature
of the postimplantation epiblast chimera assay that disallows
reinitiation of pregnancy to term, it is difficult to assess the difference between the lineage contribution. Changes to teratoma
tissue contribution do not necessarily reflect true embryonic developmental potential, but it is the best assay we currently have for
human pluripotent cells.
LIF-related receptor function is active in the naïve state, as
determined by array analysis and STAT3 inhibition. Thus, upregulation of LIFR and gp130 in the context of stem cell morphology is likely a good indicator of the naïve state in the absence of other markers. The presence of miR-302b indicates
pluripotency, whereas an increase in miR-518b, miR-520b, and
miR-520c indicates a move to an earlier pluripotency stage. Mitochondrial morphology is also a good indicator of a naïve line.
Once an appropriate morphology is detected, a few relatively
inexpensive tests of mitochondrial morphology and gene expression can detect the naïve state. Elf1 cells can be used to develop an
in vitro differentiation assay to detect the extent of endoderm
development as a naïve hallmark, and the naïve parameters above
serve as indirect evidence of developmental potential.
In summary, a stable, naïve hESC state does exist without the
need for transgenes and can be maintained indefinitely through
trypsin passage. The cells seem karyotypically stable with hallmarks consistent with a mouse naïve state, including gene expression, teratoma formation, and immature mitochondrial and
overall morphology. Naïve hESCs are capable of robust differentiation to all three germ lineages, with a heightened capability
of endoderm formation not previously seen in hESCs.
Methods
Oversight. Animals. All procedures with mice followed the recommendations
and approval of the Institutional Animal Care and Use Committee of the
University of Washington and were performed within an American Association for the Advancement of Laboratory Animal Care-accredited specific
pathogen-free facility at the University of Washington.
Human subjects. Approval was received from the University of Washington
Institutional Review Board to obtain consent for donation of excess cryopreserved human embryos from fertility clinic patients to generate new
human ESC lines. All embryo donations followed informed consent.
Embryonic stem cell research oversight. All embryonic stem cell work was preapproved by the University of Washington Embryonic Stem Cell Research
Oversight Committee.
Cell Line Nomenclature.
Cell line name “p” + number: total passage number
L: leukemia inhibitory factor
F: FGF2
A: Activin A
B/S: butyrate + SAHA HDACi mixture
2i: PD0325901 (MEK inhibitor) + CHIR99021 (GSK3 inhibitor)
Ware et al.
M: MEF conditioned medium
Parentheses: previous factor exposure
to the right of parentheses: current factor exposure
associated numbers: number of passages under those conditions
For example, H1p52(B/S3)2iF10 indicates H1 cultured for a total of 52
passages. Cells were exposed to butyrate + SAHA for three passages from
p40–p42 and then to 2i + FGF for 10 passages from p43 to p52.
Culture of Pluripotent Cells. Initial cultures of hESC [H1 (NIH code WA01), H7
(NIH code WA07), H9 (NIH code WA09), BG02, and HUES2], nonhuman primate M. fascicularis (MF-1), mEpiSC5, and mEpiSC7 ES cells were grown on
a feeder layer of gamma-irradiated (3,000 Rad) primary mouse embryonic
fibroblasts (MEFs) (7, 25). For cultures without feeders, cells were plated on
Matrigel (BD Biosciences) diluted according to the manufacturer’s instructions. Cultures were incubated at 37 °C in 5% C02, 5% O2, and 90% N2. hESC
culture medium consisted of high-glucose DMEM/F12 containing GlutaMax
supplemented with 20% KnockOut serum replacer (SR), 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 50 U/mL penicillin, 50 mg/mL
streptomycin (all from Invitrogen), and 0.1 mM β-mercaptoethanol (Sigma–
Aldrich). To reverse toggle, hESCs were first cultured in the presence of an
HDACi mixture containing 0.1 mM sodium butyrate (Sigma–Aldrich) plus
50 nM SAHA (B/S; Cayman). Human LIF was used when LIF is specified for
human and nonhuman primate ESCs. Factors were used at the following
concentrations: 10 ng/mL hLIF (Chemicon), 10 ng/mL Activin A (Humanzyme),
10 ng/mL FGF2 (Invitrogen), 0.4–0.8 μM PD0325901 (Selleck), 1.0–3.0 μM
CHIR99021 (Selleck), 2 μM SU5402 (Calbiochem), 0.1 μM NSC74859 (Selleck),
and 10 μM Y-27632 (Selleck). Primed cells and human cells in B/S were exposed
to 1.2 U/mL dispase (Invitrogen) dissolved in PBS supplemented with SR. Naïve
cells and mouse cells in B/S were passaged using trypsin/EDTA (Invitrogen).
mESCs were cultured in either DMEM or DMEM/F12 with the additives
described above, except the serum replacer was substituted with 20% FBS (ES
qualified; Invitrogen), unless otherwise stated. MEF-free cultures were grown
on Matrigel- coated dishes using medium supplemented as indicated in the
text. Mouse LIF was used at 1,000 units/mL for mESCs (ESGRO; Chemicon).
Cells for undirected differentiation were seeded at ∼2 × 105 cells per
uncoated, gelatinized, or Matrigel-treated 10-cm plate in DMEM (Invitrogen) containing 20% FBS (ES cell tested; Invitrogen) with penicillin and
streptomycin (Invitrogen). Cells were allowed to attach and fluid was
changed every other day.
5% O2 atmosphere throughout. Four days after thaw, the zona pellucida
was removed using Acidified Tyrode’s Solution and the zona-free embryo
plated into one well of a 96-well plate seeded with feeders. The first passage
was mechanical 3 d following the removal of the zona. Seven days later,
passage was through mechanical plucking of a growth above the plated
colony. Subsequent passages used Trypsin/EDTA. The culture seemed
established by passage 5. Henceforth, the cells were passaged two times per
week using trypsin/EDTA. Elf1 was bulk-frozen at passage 8 in hESC culture
medium plus 10–12 ng/mL FGF2, 3 μM CHIR 99021, and 0.4 μM PD0329501
(2iF) using the cryoprotectant dimethylsufloxide (10%; Sigma) and given the
name Elf1, “El” to honor the Ellisons, who donated the funds for the work,
and “f1” for female, line one. Elf1 is on the NIH Stem Cell Registry (0156;
approved May 15, 2012) and is being banked at WiCell for distribution.
Microarray Analysis. Whole mouse and whole human genome arrays (Agilent)
were hybridized with total RNA from ESCs cultured with additives as indicated in the text. These were run in independent quadruplicate through the
array facility at Seattle Biomedical Research Institute.
Microarray Data Preprocessing. The data for each experiment were stored as
raw, preprocessed, and processed in Synapse Commons Repository (https://
synapse.sagebase.org/). With the exception of the Hunter et al. study (8)
(GSE8881), we implemented the default one-color Affymetrix and default
two-color Agilent workflows for each dataset. The former removes intensitydependent array effects, and the latter removes both intensity-dependent
array and dye effects. A single study, the Elf1 dataset, required a more sophisticated model, which in this case meant correcting for the effects of an
outlier sample. Further methods are defined in Supporting Information.
Immunohistochemistry. Triple immunofluorescent labeling to detect endoderm in teratoma sections was performed as previously published with minor
modifications (26). Methods are further defined in Supporting Information.
Further methods for array analysis, immunohistochemistry, flow cytometry,
quantitative PCR, XIST FISH, bisulfite sequencing, teratoma formation, karyotype analysis, and electron microscopy can be found in Supporting Information.
Establishment of Elf1. Elf1 was thawed as a six- to eight-cell embryo, cultured
to expanded blastocyst in blastocyst medium (Sage), and kept in a 5% CO2,
ACKNOWLEDGMENTS. Eric Hayes and Eliza Curnow kindly provided the
M. fascicularis embryonic stem cells (MF1) for this project. We thank David
Hockenbery, Daciana Margineantu, and Scott Hansen for helpful discussions
during the progress of this work. John Stamatoyannopoulos, Richard Sandstrom, Theresa Canfield, Sandra Stehling-Sun, and Scott Hansen were helpful
in all aspects of the DNase I hypersensitivity analysis. Faria Ahmed and Abhinav
Nagulapally performed, processed, and analyzed the ChIP-seq analysis of Elf1.
David-Changsoo Kim facilitated the teratoma analysis. We are greatly indebted
to The Ellison Foundation and the State of Washington Life Science Discovery
Fund (4553677) for gift and nonfederal funds in support of this study. This
work was supported by National Institutes of Health, National Institute of
General Medical Sciences Grant P01 GM081619-01 and R01 GM097372.
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PNAS | March 25, 2014 | vol. 111 | no. 12 | 4489
DEVELOPMENTAL
BIOLOGY
3i: 2i + SU5402 (FGFR inhibitor)
T: TeSR2 medium