Transcript Quail Induced Pluripotent Stem Cells Derived

Quail Induced Pluripotent Stem Cells Derived Using
Human Reprogramming Factors
Yangqing Lu1,2,4, Franklin D. West1,2, Brian J. Jordan3, Jennifer L. Mumaw1,2, Erin T. Jordan1,2, Amalia Gallegos-Cardenas1,2, Robert B. Beckstead2, Steven L. Stice1,2
1 Regenerative
Bioscience Center, 2Department of Animal and Dairy Science, 3Department of Poultry Science, The University of Georgia, Athens GA 30602, USA
4State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Guangxi University, Nanning, Guangxi 530004, China
INTRODUCTION
As a developmental model quail-chicken chimeras have
led to the elucidation of key facets in developmental
patterning and cell fate, partially due to the ease of
access to and the ability to manipulate the avian embryo.
Avian pluripotent stem cells and derived committed cell
lines offer a cell source which could recapitulate normal
development in vitro and in vivo when transplanted into
embryos and provide the opportunity to altered
development through genetic manipulation of the stem
cells.
Characterization of qiPSC
Dapi
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OBJECTIVES
To establish a quail induced
pluripotent stem cell (qiPSC) line
that could be used in
developmental biology or
transgenic animal research.
qiPSCs demonstrate rapid proliferation, high
levels of telomerase activity and clonal
expansion after genetic manipulation
qiPSC doubling time was 16.6 hr (n=3),
significantly faster than the QEF cells (36.9 hr; P<
0.01) (A). Telomerase activity in qiPSC was higher
than QEF (>11 fold, *P<0.01) and comparable to
HeLa cells (P=0.07) (B). Eight days post
transduction a subpopulation 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).
qiPSC Produce Chimeras
Injection of qiPSC into stage X chicken embryo
To inject qiPSCs into chicken embryos, a single window was drilled into the
shell of stage X White Leghorn chicken egg (A). qiPSC were then injected
into the subgerminal cavity with a micropipette (B). Windows were sealed
with hot glue and injected eggs were then transferred to incubators (C).
qiPSCs express pluripotent genes
Immunocytochemistry demonstrated that QEFs were
negative for POU5F1 (A) and SOX2 (B), while qiPSCs
were POU5F1 (D) and SOX2 (E) positive. Scale bars
are 50 μm.
RESULTS
Derivation of qiPSC
Differentiation of qiPSC
Chimeric chicken embryos derived from qiPSC
qiPSCs generate Embryoid Bodies (EBs)
that form all 3 germ layers
Quail embryonic
fibroblast cells
(QEFs) were
isolated from day
11 embryos and
cultured in DMEM.
QEFs underwent
lentiviral transduction
utilizing viPS kit
(Thermo Scientific)
containing the human
stem cell genes POU5F1,
NANOG, SOX2, LIN28,
KLF4 and C-MYC.
Compact EBs were formed after 6 days in culture
(A). EBs were replated for further differentiation for
2 days (B). Ectoderm (TUJ1, 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 (αSMA, F) proteins (Dapi merge D’-F’).
Scale bars are 50 μm.
qiPSCs were observed
around 17 days after
transduction growing
as compact colonies.
qiPSCs were manually
harvested and plated on
Matrigel coated dishes
in mTeSR1 medium
GFP+ qiPSCs were incorporated into the brain (A, ectoderm), eye (B,
ectoderm), trachea/lung (C, endoderm), heart (D, mesoderm) and yolksac
(F, extraembryonic tissue) of quail-chicken chimeric embryos. PCR results
demonstrated that various tissues and qiPSCs were positive for the
hPOU5F1 transgene, but negative in chicken embryonic fibroblast (CEF)
(E, G).
Chimeric chickens derived from
qiPSCs
Two chicks produced by low passage
qiPSC (P7) determined to be at day 14
(A) and 19 (B) developmental stages
exhibited significant levels of feather
chimerism (black arrow), yet failed to
hatch. High passage qiPSC (P45)
contributed to live chimeras as
indicated by the presence of the human
POU5F1 gene in the brain, liver and
gonad of two individuals out of 15
examined individuals ( C).
CONCLUSION
Directed differentiation of qiPSCs to three neural
lineages.
Derivation of qiPSCs from QEFs
QEFs prior to addition of reprogramming factors (A).
Incomplete reprogrammed QEFs maintained a fibroblast-like
morphology at day 6 post-transduction (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 borders
and prominent nucleoli (D, E). qiPSCs were positive for AP (F)
and PAS (G). 5 out of 6 human pluripotent stem cell factors were
integrated and expressed in qiPSCs (H).
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, followed by
continual maintenance in differentiation medium. Neurite
extensions could be found after culturing in differentiation
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- (D) like cells after 23 and
39 days of differentiation, respectively. Scale bars in E, F and
G are 50 μm. Scale bars in D are 100 μm.
qiPSC could be derived from QEFs by
using human stem cell factors and could
generate quail-chicken chimeras and
undergo advanced in vitro differentiation.
ACKNOWLEDGEMENTS
This work was jointly supported by the Bill and
Melinda Gates Foundation and the Guangxi
Scholarship Fund.