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专业: 动物遗传学

[求助] Avian-Induced Pluripotent Stem Cells Derived Using Human Reprogramming Factors

Avian-Induced Pluripotent Stem Cells
Derived Using Human Reprogramming Factors
Yangqing Lu,
1–3
Franklin D. West,
2,3
Brian J. Jordan,
4
Jennifer L. Mumaw,
2,3
Erin T. Jordan,
2,3
Amalia Gallegos-Cardenas,
2,3
Robert B. Beckstead,
4
and Steven L. Stice2,3
Avian species are important model animals for developmental biology and disease research. However, unlike in
mice, where clonal lines of pluripotent stem cells have enabled researchers to study mammalian gene function,
clonal and highly proliferative pluripotent avian cell lines have been an elusive goal. Here we demonstrate the
generation of avian induced pluripotent stem cells (iPSCs), the ﬁrst nonmammalian iPSCs, which were clonally
isolated and propagated, important attributes not attained in embryo-sourced avian cells. This was accom-
plished using human pluripotency genes rather than avian genes, indicating that the process in which mam-
malian and nonmammalian cells are reprogrammed is a conserved process. Quail iPSCs (qiPSCs) were capable
of forming all 3 germ layers in vitro and were directly differentiated in culture into astrocytes, oligodendrocytes,
and neurons. Ultimately, qiPSCs were capable of generating live chimeric birds and incorporated into tissues
from all 3 germ layers, extraembryonic tissues, and potentially the germline. These chimera competent qiPSCs
and in vitro differentiated cells offer insight into the conserved nature of reprogramming and genetic tools that
were only previously available in mammals.
Introduction
I
n mammals, embryonic stem cells (ESCs) and now in-
duced pluripotent stemcells (iPSCs) are highly proliferative
populations capable of differentiating into multiple cell types
and tissues representing all 3 germ layers and the germline
[1–5]. Similar to ESCs,mammalian iPSCs exhibit high levels of
plasticity in both in vitro and in vivo environments [1]. After
the ﬁrst derivation of mouse iPSCs in 2006, this technology
has ﬁrmly proven its potency in generating iPSCs from vari-
ous mammals and is advancing many scientiﬁc ﬁelds, from
regenerative medicine to basic developmental biology [1,2,5–
10]. Given the diverse applications of mammalian iPSCs,
avian iPSCs would have similar utility for biologists, facili-
tating gene function, tissue transplant studies, and the rapid
generation of transgenic animals.
Although avian ESC and primordial germ cell (PGC)
lines have been established [11,12], they have not been used
in gene targeting studies, mostly likely because they have
not been clonally isolated nor are they highly proliferative
in extended cultures. Avian ESCs and PGCs have often
demonstrated a signiﬁcant decrease in their potential to
form chimeric animals after extended culture, typically < 10
passages, further reducing their potential in in vivo gene
target studies. Additionally, many of these avian lines have
not been shown to robustly undergo directed in vitro dif-
ferentiation into multiple lineages with phenotypic charac-
teristics similar to mammalian pluripotent cells. Given some
of the issues that plague the development of avian ESCs,
the probability that avian iPSCs could be generated and
show diverse differentiation potential in vitro and in vivo
seemed unlikely. Generating nonmammalian iPSCs poses
additional challenges and difﬁculties. Intuitively, species-
speciﬁc or at least species-related reprogramming factors
maybe required for proper reprogramming. However, little
is known of the required reprogramming factors in phylo-
genetically diverse species. Previous studies showed that
iPSC could be derived from human, mouse, pig, primate,
and even rhinoceros ﬁbroblast using the human repro-
gramming factors [5,6,13,14]. These results indicated the
widely conserved reprogramming process among species
and the possibility of deriving iPSCs from phylogenetically
diverse species such as avian.
Avian embryonic models have a long history of provid-
ing critical new insights into developmental biology in-
cluding organ function [15,16], disease progression (e.g.,
Pompe disease) [17], eye disorders [18], and many others
[19,20]. The advantage of avian species is their relative size
1
State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Animal Reproduction Institute, Guangxi
University, Nanning, Guangxi, China.
2
Regenerative Bioscience Center, University of Georgia, Athens, Georgia.
Departments of
3
Animal and Dairy Science and 4
Poultry Science, University of Georgia, Athens, Georgia.
STEM CELLS AND DEVELOPMENT
Volume 21, Number 3, 2012
Mary Ann Liebert, Inc.
DOI: 10.1089/scd.2011.0499
394and ease of access to the embryo for manipulation. Cells
and tissues, including whole sections of the spinal column,
can be transplanted into the avian embryo and can be
monitored in real time during development [21]. This is not
possible in mammalian species.Moreover, the quail-chicken
chimera is an attractive and widely used model for devel-
opmental patterning and cell fate studies given that cells
canbereadilytrackedinthismodel[22,23].Thequailalso
has a short generation interval (3–4 generations per year)
[24], facilitating genetic selection studies and experiments
requiring multiple generational observations [25]. Robust
clonal and feeder-free iPSC lines capable of generating
lineage-committed avian cells offer new opportunities to
manipulate and study developmental process both in vitro
and in vivo.
In this report we demonstrate for the ﬁrst time in a non-
mammalian species the successful generation of a highly
proliferative quail iPSC (qiPSC) population amenable to ge-
netic manipulation, that undergoes advanced directed neu-
ral differentiation in vitro and is capable of generating live
chimeric offspring. The extensive contribution of qiPSCs to
chimeric animals even at late passages demonstrates that
thesecellsaretrueiPSCs.TheseﬁrstiPSCsinanonmammalian
species demonstrate the highly conserved nature of the repro-
gramming genes and provide for future mechanistic develop-
mental studies in well-characterized avian models.
Materials and Methods
Cell culture and transduction
Quail embryonic ﬁbroblasts (QEFs) were isolated from
day-11 embryos and cultured in ﬁbroblast medium [Dul-
becco’s modiﬁed Eagle’s medium (DMEM) high glucose
(Hyclone) with 10% fetal bovine serum (Hyclone), 4mM L-
glutamine (Gibco), and 50U/mL penicillin and 50 mg/mL
streptomycin (Gibco)] in 5% CO2 at 37C. Cells were split
using 0.05% trypsin (Gibco) upon reaching conﬂuence.
For transduction, a total of 150,000 QEF cells was plated in
1 well of a 12-well plate. After 24 h, QEFs underwent len-
tiviral transduction utilizing the viPS kit (Thermo Scien-
tiﬁc) with viruses containing the human stem cell genes
POU5F1, NANOG, SOX2, LIN28, KLF4,and C-MYC under
the promoter of human elongation factor-1 alpha (EF1-a).
Transduction was performed in the presence of 1 ·
TransDux (System Biosciences). The QEFs were trypsinized
24 h after transduction and passaged onto inactivated fee-
der cells in ESC expansion medium [DMEM/F12 (Gibco),
supplemented with 20% knockout serum replacement
(KSR; Gibco), 2mM L-glutamine (Gibco), 0.1mM nones-
sential amino acids (Gibco), 50U/mL penicillin/50 mg/mL
streptomycin (Gibco), 0.1mM b-mercaptoethanol (Sigma-
Aldrich), and 10 ng/mL basic ﬁbroblast growth factor
(bFGF; Sigma-Aldrich and R&D Systems)]. The qiPSCs
were manually harvested and plated on Matrigel (BD
Biosciences; diluted 1:100 in DMEM/F12) coated dishes in
mTeSR1 medium (Stemcell Technologies). The qiPSCs were
mechanically dissociated using a glass Pasteur pipette
every 4–5 days. For clonal expansion, qiPSCs were trans-
duced with green ﬂuorescent protein (GFP) viral vector
and single cells were FACS sorted into individual wells of a
96-well plate.
Alkaline phosphatase and periodic acid
Schiff’s staining
Alkaline phosphatase (AP) staining was carried out with
VECTOR Red Alkaline Phosphatase Substrate Kit (Vector
Laboratories) according to the manufacturer’s instructions.
Periodic acid Schiff (PAS) staining was performed by 4%
paraformaldehyde ﬁxation for 5min. The PAS (Sigma-
Aldrich) was added to the plate and incubated at room tem-
perature for 5min, followed by phosphate-buffered saline
(PBS; Hyclone) washes for 3 times. Schiff’s reagent (Sigma-
Aldrich) was added and incubated at room temperature for
15min, followed by 3 washes with PBS and then observation.
Immunocytochemistry
Protocol for immunostaining followed the methods pre-
viously reported [6]. Primary antibodies used were POU5F1
(R&D Systems), SOX2 (R&D Systems), TUJ1 (Neuromics),
alpha smooth muscle actin (aSMA) (Santa Cruz), SOX17
(Santa Cruz), SSEA4 (Developmental Studies Hybridoma
Bank), TRA-1-60 (Chemicon), TRA-1-81 (Chemicon),Hu C/D+
(Invitrogen), MAP2 + (Millipore), GFAP (Chemicon), and O4
(Chemicon). Secondary antibodies used in this study were all
from Invitrogen. Cell observations and images were captured
on Ix81 with Disc-Spinning Unit (Olympus) using Slide Book
Software (Intelligent Imaging Innovations).
Proliferation and telomerase activity
Proliferation assay was performed by manual counts
(n = 3) at 12, 24, 36, and 48 h after plating. Population dou-
bling time was determined using an exponential regression
curve ﬁtting (www.doubling-time.com/compute.php). Tel-
omerase activity of QEFs, qiPSCs, and HeLa cells (positive
control) was determined using TRAPeze XL Telomerase
Detection Kit (Millipore) following the manufacturer’s in-
structions. Statistical analysis was done utilizing ANOVA
and Tukey pair-wise comparisons between each population
with 2-tailed P value < 0.05 being considered signiﬁcant.
Embryoid body formation and differentiation
Embryoid bodies (EBs) were formed by plating 2.0 · 106
qiPSCs in mTeSR1 medium and 0.1mM Y-27632 ROCK in-
hibitor (Calbiochem) in an AggreWell plate (Stemcell Technol-
ogies). After 24 h, aggregateswere harvested andmaintained in
mTeSR1 medium for 7 days. Differentiation was assessed by
reverse transcription polymerase chain reaction (RT-PCR) using
the primers in Supplementary Table S1 (Supplementary Data
are available online at www.liebertonline.com/scd). To assess
the differentiation by immunocytochemistry, EBs were plated
on 4-chamber slides and fed with 20% KSR media without
bFGF, which allowed a further differentiation for 2 days.
Neural differentiation
To induce neural differentiation, qiPSCs were sequentially
cultured in neural derivation medium [DMEM/F12 supple-
mented with 200mM L-glutamine, 4 ng/mL bFGF, and 1 ·N2
(Gibco)] for 12 days, proliferation medium [AB2 (Aruna)
medium supplemented with 200mM L-glutamine, 1 · aruna
neural supplement (ANS), and 20 ng/mL bFGF] for 7 days, and
QUAIL IPSC GENERATED BY HUMAN REPROGRAMMING FACTORS 395then in differentiation medium (AB2 medium supplemented
with 200mM L-glutamine, 1 ·ANS, and 10 ng/mL leukemia
inhibitory factor [LIF]) continuously.
Production of chimera
Stage X White Leghorn chicken embryos were used to pro-
duce chimeras. Egg shells were removed with a Dremel rotary
tool to make an injection window (Supplementary Fig. S2A).
The qiPSCs were introduced into the subgerminal cavity using
a glass micropipette (Supplementary Fig. S2B) with pressure
controlled microinjector (Parker Automation). Cells were pre-
pared at concentration of 10,000,000 per mL and injected 1 mL
per embryo, which should be approximately 10,000 cells. The
windowwas sealed by hot glue (Supplementary Fig. S2C) after
injection and eggs were incubated at 37.8C.
RNA isolation, DNA isolation, PCR, and sequencing
RNA was isolated using RNeasy QIAprep Spin miniprep
Kit (Qiagen) per manufacturer’s instructions. Genomic DNA
was removed using gDNA eliminator columns (Qiagen).
mRNA extractions were transcribed into cDNA using iScript
cDNA Synthesis kit (Bio-Rad Laboratories). DNA was iso-
lated using DNeasy kit (Qiagen) following the manufactur-
er’s instructions. Primers used in PCR and RT-PCR are listed
in Table S2. Sequencing veriﬁcation of hPOU5F1 was per-
formed by extracting DNA from agarose gels after electro-
phoresis and sequencing. The resulted sequence was
compared by Blast in the NCBI database to both human and
chicken genomes.
Results
qiPSCs display morphological characteristics
consistent with a pluripotent cell type
The generation of qiPSCs was initiated by testing the
lentiviral transduction efﬁciency of isolated QEF (Fig. 1A)
with an eGFP reporter construct using both GeneJammer
and TransDux transduction reagents. A 20 multiplicity of
infection (MOI) transduction with Transdux resulted in the
FIG. 1. Derivation of qiPSCs from
QEFs. QEFs prior to addition of re-
programming factors (A). In-
complete reprogrammed QEFs
maintained a ﬁbroblast-like mor-
phology at day 6 posttransduction
(B), while qiPSC colonies at day 17
showed deﬁned borders (C) and at
the single-cell level, a high nuclear-
to-cytoplasm ratio, clear cell bor-
ders, and prominent nucleoli (D, E).
qiPSCs were positive for AP (F) and
PAS (G). Five out of 6 human plu-
ripotent stem cell factors were inte-
grated and expressed in qiPSCs (H).
Immunocytochemistry demonstrated
that QEFs were negative for POU5F1
and SOX2 (I–K), while qiPSCs were
POU5F1 and SOX2 (L–N) positive.
Scale bars, 100 mm (A–F);200 mm (G);
50 mm (I–N). AP, alkaline phospha-
tase; PAS, periodic acid Schiff
staining; QEF, quail embryonic ﬁ-
broblasts; qiPSCs, quail-induced
pluripotent stem cells. Color images
available online atwww.liebertonline
.com/scd
396 LU ET AL.highest efﬁciency with 40.5% green ﬂuorescent protein
(GFP)–positive cells (Supplementary Fig. S1). QEFs were
then transduced with the 6 human pluripotency genes
hPOU5F1, hNANOG, hSOX2, hLIN28, hC-MYC, and hKLF4
driven by the EF1-a promoter with each construct in indi-
vidual lentiviral vectors. After 24 h, cells were replated on
feeder cells in stem cell expansion medium.
Colonies began to emerge 6 days after transduction with
irregular-shaped borders and ﬁbroblast-like cell morphol-
ogy (Fig. 1B). These initial colonies failed to proliferate
and expand, indicating that these colonies were not fully
reprogrammed. Potential qiPSCs were observed around 17
days after transduction and grew as compact colonies (Fig.
1C). The compact colonies were mechanically picked and
initially replated on feeder plates in stem cell expansion
medium. However, replated cells failed to proliferate and
appeared apoptotic. Additional colonies were collected and
replated on Matrigel-coated plates in mTeSR1 stem cell
medium. This system supported the growth and expansion
of colonies and subsequent qiPSC expansion was performed
using this system.
Morphologically, qiPSC colonies were highly refractive
and at the single-cell level they showed clear cell borders,
high nuclear-to-cytoplasm ratio, and prominent nucleoli (Fig.
1D, E). The qiPSCs were strongly positive for AP and PAS
staining (Fig. 1F, G). PCR and RT-PCR using human-speciﬁc
primers revealed that 5 out of 6 pluripotent stem cell factors,
hPOU5F1, hSOX2, hNANOG, hLIN28, and hC-MYC, were
integrated and expressed in qiPSCs, while hKLF4 was not
present (Fig. 1H). Immunocytochemistry revealed that
POU5F1 and SOX2 proteins were absent in QEFs (Fig. 1I–K),
but positive in qiPSCs (Fig. 1L–N). Immunocytochemistry of
qiPSCs showed that cells were negative for the pluripotency
markers SSEA4, TRA-1-81, or TRA-1-60.
qiPSCs are highly proliferative, express pluripotent
markers, and are capable of clonal expansion
after genetic manipulation
Rapid proliferation and high levels of telomerase activity
are hallmarks of pluripotent stem cells. To determine the
doubling time, plated cells were quantiﬁed every 12 h for 48 h.
The population doubling time of qiPSCs was 16.6 h, much
faster than the QEF parent cell line (36.9 h, P < 0.01) (Fig. 2A).
The qiPSCs are highly proliferative and are passaged every 4
days. Cells have been maintained for more than 50 passages
without loss of the pluripotent phenotype. Telomerase activity
revealed a signiﬁcant (P < 0.01) increase of > 11-fold from 8.4
total product generated (TPG) in QEFs to 95.3 TPG in qiPSCs
(Fig. 2B). Telomerase activity of qiPSCs was comparable to
that of the positive control HeLa cell line (50.8 TPG, P = 0.07),
which indicates the immortality of qiPSCs. Moreover, these
cells were capable of clonal expansion after genetic manipu-
lation. The qiPSCs were transduced with the eGFP gene, re-
sulting in a 36.1% GFP + population (Fig. 2C–E). Single GFP +
cells were FACS sorted into each well of a 96-well plate and
colonies were found in 2 wells 9 days after sorting (Fig. 2F, G).
Flow cytometry analysis of cells expanded from 1 colony
showed that > 96% of the cells still expressed GFP after serial
subculture (Fig. 2H).
EB differentiation of qiPSCs resulted in the formation
of all 3 germ layers
To derive EBs, qiPSCs were plated in AggreWell plates for
24 h and then transferred to suspension culture in mTeSR1
medium for differentiation. Six days of suspension culture
resulted in round and compact EBs from qiPSCs (Fig. 3A).
The EBs were collected for RNA isolation and RT-PCR or
FIG. 2. qiPSCs demonstrate rapid prolifer-
ation, high levels of telomerase activity, and
clonal expansion after genetic manipulation
qiPSC doubling time was 16.6 h (n = 3), sig-
niﬁcantly faster than the QEF cells (36.9 h;
P < 0.01) (A). Telomerase activity in qiPSCs
was higher than QEFs ( > 11-fold, *P < 0.01)
and comparable to HeLa cells (P = 0.07) (B).
Eight days posttransduction, a subpopula-
tion of qiPSCs expressed GFP (C, D, E). Nine
days after FACS sorting, clonally expanded
GFP + qiPSCs generated colonies (F, G) that
maintained GFP expression long term (H).
Scale bars, 200 mm (C, D, F, G).
QUAIL IPSC GENERATED BY HUMAN REPROGRAMMING FACTORS 397replated for additional differentiation for 2 days in stem cell
expansion medium without bFGF—the removal of which
will drive differentiation (Fig. 3B). Results of RT-PCR
showed expression of TUJ1 (ectoderm), PAX6 (ectoderm),
Vimentin (endoderm), and Brachyury (mesoderm) in EBs, but
not in qiPSCs or QEF cells (Fig. 3C). Immunocytochemistry
showed cells positive for TUJ1 (ectoderm, Fig. 3D), SOX17
(endoderm, Fig. 3E), and alpha smooth muscle actin (aSMA,
mesoderm, Fig. 3F) in plated EBs. These results indicated
that qiPSCs could differentiate into various cell types from
all 3 germ layers.
qiPSC differentiate in vitro into neurons,
astrocytes, and oligodendrocytes
To derive neural cells, qiPSCs were subjected to a 3-step
neural differentiation process. Cells were initially cultured in
neural derivation medium for 12 days, proliferation medium
for 7 days, and neural differentiation medium continuously
thereafter. Immunostaining showed that these cells were
positive for the neural proteins Hu C/D+ and MAP2 + (Fig.
4A, B) after 10 days of differentiation. A signiﬁcant number
of neurite extensions was observed after differentiation.
Differentiated qiPSCs were found to be positive for the as-
trocyte- and oligodendrocyte-associated proteins GFAP and
O4 after 23 and 39 days of differentiation, respectively, in
neural differentiation medium (Fig. 4C, D). These data
demonstrated the neural competence of qiPSC and differ-
entiation into all 3 neural lineages, which has not been seen
in any previous avian stem cell lines.
Incorporation of qiPSCs into chimeric embryo
and generation of live offspring
To generate qiPSC-chicken chimeras, GFP + qiPSCs at
passage 26 were injected into the subgerminal cavity of stage
X embryos (Supplementary Fig. S2). Embryos were incu-
bated for 14 or 19 days and were then dissected to determine
GFP + qiPSC incorporation into embryos. GFP + qiPSCs
were incorporated in brain (Fig. 5A), eye (Fig. 5B), trachea/
lung (Fig. 5C), heart (Fig. 5D), and yolk sac (Fig. 5F) tissues.
PCR was performed for the human POU5F1 gene that was
used to reprogram QEFs into iPSCs to further determine
qiPSC contribution in chimeric animals. The qiPSCs were
present in tissues from the ectoderm (brain, eye, and skin),
endoderm (intestine, liver, and lung), mesoderm (muscle and
heart), extraembryonic tissue (yolk sac), and the gonad (Fig.
5E, G and Supplementary Table S2). PCR products from
qiPSCs from the yolk sac and skin were sequenced to vali-
date that PCR primers were solely expanding the human
POU5F1 sequence. Blast of the sequenced DNA ampliﬁed
from these tissues showed identity of 99% to 100% for hu-
man POU5F1 genomic context sequence NC_000006.11, but
only 72% to 74% maximum identity for chicken PouV (Oct4
homologue) mRNA NM_001110178.1 and no signiﬁcant
similarity to its genomic context sequence NW_001471503.1.
These results indicate that qiPSCs incorporated and con-
tributed to chicken embryonic tissues from all 3 germ layers,
extraembryonic tissues, and potentially the germline.
Two additional rounds of injections were performed to
generate chimeric chicks using passage 7 and passage 45
qiPSCs. Early passage qiPSCs were injected into 30 eggs.
Three embryos developed to term and hatched after 21 days
of incubation, while 9 were unfertilized and 18 embryos failed
to hatch. Hatched offspring were not chimeric, but 2 chicks at
late stages of embryonic development showed feather chi-
merism (Fig. 6A, B). Passage 45 qiPSCs were injected into 102
eggs, resulting in 47 live offspring. Two chicks died soon after
hatching and the rest of 45 chicks were healthy, but no feather
chimerism was observed. Brain, muscle, liver, and gonad
tissues were collected from 15 chickens for PCR detection of
qiPSC incorporation. Results showed that brain, liver, and
gonad samples from 2 individuals were positive for the hu-
man POU5F1 reprogramming gene (Fig. 6C). The presence of
FIG. 3. qiPSCs generate EBs that
form all 3 germ layers. Compact EBs
were formed after 6 days in culture
(A). EBs were replated for further
differentiation for 2 days (B). Ecto-
derm (TUJ1 and PAX6), endoderm
(Vimentin), and mesoderm (Brachyury)
genes were expressed in EBs (C).
Immunocytochemistry demonstrated
that EB-derived cells were positive for
ectoderm (TUJ1, D), endoderm
(SOX17, E), and mesoderm (aSMA, F)
proteins (DAPI merge D¢–F&cent

. Scale
bars, 200 mm (A, B);50 mm (D–F, D¢–
F&cent

. EB, embryoid body.
398 LU ET AL.feather chimerism with early passage cells and incorporation
of qiPSCs into tissue with later passages indicate that these
cells retain pluripotent characteristics following long-term
culture and are still capable of contributing to multiple line-
ages.
Discussion
iPSCs have been generated from numerous mammalian
species [1–2,5–10], but never before in a nonmammalian
species. Here we report the ﬁrst nonmammalian iPSCs and
paradoxically these avian iPSCs were generated using hu-
man reprogramming factors. These chimera competent cells
were capable of producing live offspring, achieved previ-
ously only in mice, rats, and pigs, and showed signiﬁcant
incorporation into all 3 germ layers, marking their true
pluripotent nature [26]. Beyond contribution to chimeric
offspring, mouse pluripotent cells are relatively easy to ge-
netically modify and clonally isolate thus facilitating the
generation of gene-targeted offspring [27]. The qiPSCs were
also genetically manipulated and then clonally selected, as
proliferative colonies. These in vitro characteristics will likely
lead to site-directed gene insertion and selection of clonal
colonies for generating chimeras. The qiPSCs show mor-
phology consistent with previously established pluripotent
stem cells at the single-cell level. The qiPSCs are highly
positive for the stem cell markers AP, PAS, POU5F1, and
SOX2 that have been previously used to characterize avian
ESCs and PGCs [28–32]. These iPSCs are highly proliferative
with a doubling time of 16.6 h, similar to iPSCs from mouse
[1] and pig [33]. In the chick/quail chimera model, qiPSCs
contributed to fetal tissues from all 3 germ layers and ex-
traembryonic tissues and ultimately contributed to tissues in
live offspring. Although avian ESCs and PGCs have gener-
ated chimeric offspring [29,34], the qiPSCs differ because for
the ﬁrst time an avian stem cell exhibits the robust in vitro
proliferative and clonal attributes needed for future gene-
targeted birds.
In this study, qiPSCs were generated by transducing QEFs
with 6 reprogramming factors and 5 of them (POU5F1,
NANOG, LIN28,and C-MYC) were found incorporated into
the genomic DNA and expressed in high level, which indi-
cated their essential roles in the reprogramming process. KLF4
was not detected in qiPSCs in DNA or RNA level. In previous
reports, KLF4 has been shown to be important in the repro-
gramming process [1,5,35,36]. However, many groups have
shown successful reprogram in cells of multiple species
without the KLF4 gene, demonstrating that it is not essential
[2,37]. The qiPSCs generated without ectopic KLF4 showed
robust pluripotency and were capable of chimera formation,
indicating ectopic expression of KLF4 is not required in cell
reprogramming in avian species. The successful use of human
reprogramming factors to generate avian iPSCs suggests that
direct reprogramming mechanisms are widely conserved
among species. In the chicken, the cPouV and cNanog genes
were found to be key factors in the maintenance of chicken
pluripotency [38]. However, the homology of reprogramming
genes between avian and human genomes is relatively low
when comparing chicken—the only avian species with se-
quence data—and humans with homology ranging from
*53% to 81% for POU5F1, NANOG, LIN28, C-MYC,and
KLF4. SOX2 is the lone exception with homology of 94% be-
tween chicken and human genes. A previous report compar-
ing the embryonic pluripotency gene-regulatory networks
between mouse and chicken revealed that the mouse core
pluripotency network is mediated largely by genomic se-
quence elements that are not conserved within the chicken
[39]. This report suggests that species-speciﬁc or at least spe-
cies-related reprogramming factors would be required for
proper reprogramming of avian cells. However, based on the
diverse number of species (mouse, human, pig, primate, and
rhinoceros) [5,6,13,14] from which cells have been repro-
grammed using human factors and now a nonmammalian
species, we hypothesize that the reprogramming process is
highly conserved. Therefore, direct reprogramming with
transcriptional factors could be a universal strategy for gen-
erating iPSC lines in distantly related species. This would
provide new species iPSCs for divergent species including
FIG. 4. Directed differentiation of qiPSCs to 3 neural line-
ages. qiPSCs were subjected to a 3-step neural differentiation
process, with cells ﬁrst cultured in neural derivation medium
for 12 days, then in proliferation medium for 7 days, fol-
lowed by continual maintenance in differentiation medium.
Neurite extensions could be found after culturing in differ-
entiation medium for 48 days. Neuron-like cells expressing
Hu C/D+ (A) and MAP2 (B) were present after 10 days of
differentiation and astrocyte- (C) and oligodendrocyte-like
cells (D) after 23 and 39 days of differentiation, respectively
(DAPI merge A¢–D&cent

. Scale bars, 50 mm (A–D, A¢–D&cent

.
QUAIL IPSC GENERATED BY HUMAN REPROGRAMMING FACTORS 399species where ESCs are hard to isolate, maintain, and expand,
which arguably would include all species other than those of
primates and some rodents.
Quail and quail-chick chimeras have long been used in
understanding the development of the nervous system [40–
43]. With the beneﬁt of this avian system, numerous facets of
neural development in the brain [15,40] and neural crest [41]
have already been deciphered. In the present study, qiPSCs
were found to signiﬁcantly contribute to the brain and eye
tissue when injected into stage X chicken embryos in an
undifferentiated state. Upon proper signaling in vitro,
qiPSCs could differentiate into a neural progenitor (TUJ1 + )
and all 3 lineages of neural cells—neuronal (Hu C/D+ and
MAP2 + ), astrocyte (GFAP + ), and oligodendrocyte (O4 + )—
in vitro. Despite preliminary neural differentiation of chicken
ESCs or embryonic germ cell into neural cell [11,12], the
desired diversity in neural developmental competence,
forming neuronal both glia lineages, (TUJ1, MAP2, Hu C/D,
GFAP, and O4) was not present or reported [44–46]. Given
that we have derived neurons, astrocytes, and oligodendro-
cytes using processes ﬁrst developed in human pluripotent
cells [47], the repertoire of ‘‘tools’’ and translational potential
for embryonic graft studies is greatly enhanced. Temporal
and spatial studies investigating the role and interaction
among neural cell types during embryonic development are
now possible. Pluripotent qiPSC-derived neural cells can be
compared with tissues from fetal and adult organisms faster
and in relevant but comparatively simple models in contrast
to mammalian systems [21].
The methods used here for qiPSCs overcome impedi-
ments inherent to avian ESCs and PGCs. Previously quail or
FIG. 5. Chimeric chicken embryos de-
rived from qiPSCs. GFP+ qiPSCs were
incorporated into the brain (A, ectoderm),
eye (B, ectoderm), trachea/lung (C, endo-
derm),heart (D, mesoderm), and yolk sac
(F, extraembryonic tissue) of quail-chicken
chimeric embryos. Polymerase chain reac-
tion results demonstrated that various
tissues and qiPSCs were positive for the
hPOU5F1 transgene, but negative in chick-
en embryonic ﬁbroblast (CEF) (E, G) (GFP
andbrightﬁeldmerge A¢–D&cent

. Color ima-
ges available online at www.liebertonline
.com/scd
FIG. 6. Chimeric chickens derived from qiPSCs. Two chicks
produced by low-passage qiPSCs (P7) determined to be at
day 14 (A) and 19 (B) developmental stages exhibited sig-
niﬁcant levels of feather chimerism (black arrow), yet failed to
hatch. High-passage qiPSCs (P45) contributed to live chi-
meras as indicated by the presence of the human POU5F1
gene in the brain, liver, and gonad of 2 individuals out of 15
examined individuals (C). Color images available online at
www.liebertonline.com/scd
400 LU ET AL.chicken PGCs [32,34] and ESCs [29] contributed to chimeras
when injected into embryos immediately after collection
from the donor embryo or after only a few passages. After
serial subculture in this system, qiPSCs still efﬁciently in-
corporated into tissues from all 3 germ layers in chimeric
embryos at passages 26 and 45. This will further enable
complex genetic manipulations like homologous recombi-
nation,multiplegeneintroductions, drug selection, and
other strategies that may require extended culture. This
signiﬁcantly increases the value of these cells for future
developmental studies. In addition, cultures beyond 50
passages that maintain a short doubling time and a plu-
ripotent phenotype have not been previously reported.
Beyond long-term culture, qiPSCs demonstrated the capa-
bility of clonal expansion after isolating individual cells in a
96-well format, providing the possibility of targeting genes
of interest. Little is known about the maintenance of plur-
ipotency and expansion of avian pluripotent cells. There-
fore, we used methods based on the signiﬁcant body of
knowledge for mammalian pluripotent cells [48–52]. The
feeder-free culture system that supported qiPSC cultures for
more than 50 passages without loss of the pluripotent
phenotype was developed for human pluripotent cells and
contains high levels of bFGF [53]. In the future, this system
can be used to investigate individual factors and their role
in maintaining pluripotency of avian stem cells. The qiPSC
gene-targeted avian models will compliment important
rodent models in disease and developmental gene function
studies [27].
We report here the availability of chimera competent
avian iPSCs that will greatly facilitate gene targeting and the
insertion of genetic reporters. Future studies that generate
cells with gene-speciﬁc and multiple promoters, inducers,
and conditional expression systems in avian iPSCs are fea-
sible, thus enhancing the research community’s capabilities
when it comes to directly observing cell migration and em-
bryonic and fetal development in ova [54]. Since these
qiPSCs were capable of clonal expansion after genetic mod-
iﬁcation, targeting genes of interest is potentially possible,
which would facilitate research on gene function and sig-
naling pathways underlying the development process in
chimeric embryos. Further, avian iPSC derivatives such as
neural cells should compliment mammalian cell transplant
models for regenerative medicine [55]. In total, this unique
source of avian iPSCs and derivative cells will provide bi-
ologists with multiple opportunities to enhance and expedite
developmentally related discoveries.
Acknowledgments
The authors thank Dr. Jeanna L. Wilson (Department of
Poultry Science, University of Georgia) for providing quail
embryos, Anand Subramanian (Department of Animal and
Dairy Science, University of Georgia) for immunostaining of
neural marker MAP2, and Julie Nelson at the Center for
Tropical and Emerging Global Diseases Flow Cytometry
Facility. This work was jointly supported by the Bill and
Melinda Gates Foundation and the Guangxi Scholarship
Fund.
Author Disclosure Statement
No competing ﬁnancial interests exist.
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Address correspondence to:
Prof. Steven L. Stice
Regenerative Bioscience Center
University of Georgia
425 River Road, ADS
Athens, GA 30602
E-mail: sstice@uga.edu
Received for publication September 1, 2011
Accepted after revision October 3, 2011
Prepublished on Liebert Instant Online October 4, 2011
QUAIL IPSC GENERATED BY HUMAN REPROGRAMMING FACTORS 403

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