Human Amniotic Mesenchymal Stem Cell-Derived
Induced Pluripotent Stem Cells May Generate
a Universal Source of Cardiac Cells
Xiaohu Ge,1,*I-Ning E. Wang,1,*Ildiko Toma,1Vittorio Sebastiano,2Jianwei Liu,3
Manish J. Butte,3Renee A. Reijo Pera,2and Phillip C. Yang1
Human amniotic mesenchymal stem cells (hAMSCs) demonstrated partially pluripotent characteristics with a
strong expression of Oct4 and Nanog genes and immunomodulatory properties characterized by the absence of
HLA-DR and the presence of HLA-G and CD59. The hAMSCs were reprogrammed into induced pluripotent
stem cells (iPSCs) that generate a promising source of universal cardiac cells. The hAMSC-derived iPSCs
(MiPSCs) successfully underwent robust cardiac differentiation to generate cardiomyocytes. This study inves-
tigated 3 key properties of the hAMSCs and MiPSCs: (1) the reprogramming efficiency of the partially plurip-
otent hAMSCs to generate MiPSCs; (2) immunomodulatory properties of the hAMSCs and MiPSCs; and (3) the
cardiac differentiation potential of the MiPSCs. The characteristic iPSC colony formation was observed within 10
days after the transduction of the hAMSCs with a single integration polycistronic vector containing 4 Yamanaka
factors. Immunohistology and reverse transcription–polymerase chain reaction assays revealed that the MiPSCs
expressed stem cell surface markers and pluripotency-specific genes. Furthermore, the hAMSCs and MiPSCs
demonstrated immunomodulatory properties enabling successful engraftment in the SVJ mice. Finally, the
cardiac differentiation of MiPSCs exhibited robust spontaneous contractility, characteristic calcium transience
across the membrane, a high expression of cardiac genes and mature cardiac phenotypes, and a contractile force
comparable to cardiomyocytes. Our results demonstrated that the hAMSCs are reprogrammed with a high
efficiency into MiPSCs, which possess pluripotent, immunomodulatory, and precardiac properties. The MiPSC-
derived cardiac cells express a c-kit cell surface marker, which may be employed to purify the cardiac cell
population and enable allogeneic cardiac stem cell therapy.
implications in regenerative medicine. Many strategies have
been developed for iPSC generation, including genomic in-
tegration, synthetic mRNA, small molecules, and protein-
optimalcell population, which canbe readilyinduced intothe
pluripotent state, may be equally important. More notewor-
thy is that the current iPSC reprogramming strategy is an
inefficient and slow process, which may limit their immediate
usage in biological and translational research . Differ-
entiated cells are known to demonstrate lower reprogram-
ming efficiency, and different somatic cells are found to
possess differential reprogramming ability . In human fi-
he generation of induced pluripotent stem cells
Yamanaka’s factors (Sox2, Klf4, Oct4, cMyc; SKOM) form
AP+ (alkaline phosphatase) iPSC colonies [7–9]. The robust
and rapid generation of iPSCs has raised an important chal-
lenge in the field of stem cell research and regenerative
medicine. In this study, we report a unique population of the
human amniotic mesenchymal stem cells (hAMSCs) with a
high reprogramming efficiency to generate iPSCs.
Placental tissue is readily available, easily procured
without invasive procedures, and does not elicit ethical
debate. Two regions of the amniotic membrane of the pla-
centa contain the partially pluripotent epiblast population
of the human amniotic epithelial cells and extraembryonic
mesoderm population of hAMSCs . These cells have
been described as differentiating predominantly along
the mesodermal lineage and as demonstrating precardiac
1Division of Cardiovascular Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, California.
2Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, California.
3Division of Pediatrics Immunology & Allergy, Stanford University School of Medicine, Stanford, California.
*These two authors contributed equally to this work.
STEM CELLS AND DEVELOPMENT
Volume 21, Number 15, 2012
? Mary Ann Liebert, Inc.
commitment [11–13]. Furthermore, recent reports indicate
partial pluripotency of the hAMSCs with a high expression
of pluripotency-specific genes, Nanog and Oct4 . In
addition, the hAMSCs demonstrate the immunomodula-
tory properties that are known to suppress host immune
responses. Interestingly, amniotic cells have never shown
signs of aging and tumorigenecity even after propagation
for more than 2 years in culture .
The hAMSCs were transduced via polycistronic lentivirus
containing 4 transcription factors: Oct4, Sox2, c-Myc, and
Klf4. The hypothesis that the robustly generated hAMSC-
derived iPSCs (MiPSCs) will exhibit immunomodulatory
and cardiac differentiation properties was tested. The find-
ings from this study demonstrated that the hAMSCs gener-
ate a robust population of iPSCs (MiPSCs) characterized by
stem cell surface markers, pluripotency genes, and immu-
nomodulatory properties. More significantly, the MiPSCs
readily demonstrated spontaneous contractility on day 12 of
the cardiac differentiation protocol with mature cardiac
phenotypes. This study suggests that these characteristics of
MiPSCs may enable a source of universal cardiac cells.
Materials and Methods
hAMSC isolation from the human placenta
Human placentas were obtained from healthy subjects at
the Stanford University Medical Center, Stanford, CA. All
donors provided written informed consent before collection.
Under stringent sterile conditions, the harvested placentas
were placed in HBSS media (Invitrogen). The human amni-
otic membrane was carefully separated from the chorion, and
the membrane was immediately washed 3 to 5 times with
0.9% NaCl solution to remove blood and mucus. The mem-
brane was cut into 2·2cm pieces and transferred into an
enzymatic digestion buffer containing trypsin-EDTA (In-
vitrogen) in phosphate-buffered saline and incubated at 37?C
for 10, 20, and 30min. The digested tissue was centrifuged,
and the supernatant was discarded. Then, the tissue was
subjected to a second enzymatic digestion in 50mL HBSS
(Invitrogen) containing 50mg type I collagenase (Invitrogen),
0.01% papain (Sigma), and 10% fetal bovine serum (FBS) for
2h at 37?C. After digestion, the suspension was filtered
through a sterile 70mm filter (BD Biosciences), and the cells
were collected by centrifugation at 200 g for 5min. The col-
lected cells were designated as hAMSCs. The sorted cells
were cultured in the Dulbecco’s modified Eagle medium
(DMEM) supplemented with 110mg/L sodium pyruvate,
4mM l-glutamine, 10% FBS, 1% Pen-Strep, and 10ng/mL
EGF (R&D Systems) at 37?C, 5% CO2.The c-kit (+) sub-
population of hAMSCs was fluorescent activated cell sort
(FACS) sorted by excluding the HLA-DR, CD34, and CD45
(immunity and hematopoietic markers; Biolegend) cells and
by including the SSEA3, SSEA4, TRA1-60, TRA1-81, thy-1,
and c-kit (stemness and cardiac markers; Biolegend) cells.
Virus production and iPSCs generation
The plasmid of pHAGE2-EF1a-OKSM vectors (courtesy of
Mostoslavsky, G, Ph.D., Boston University) was employed.
To generate the virus, 293T cells were transfected at 90%
confluence using lipofectamine (Invitrogen). For a 10cm
plate, 1mL OPTIMEN (Invitrogen), 36mL lipofectamine, and
24mg DNA mixture (20:1:1:1:2; pHAGE2: tat: rev: gag/pol:
vsv-g) were used. The virus was harvested over 3 days and
concentrated by spinning for 1.5h at 16,500 RPM at 4?C.
Approximately, 100,000 hAMSCs were seeded using a 6-well
plate and infected with 150mL of the concentrated virus in the
presence of polybrene (10mg/mL; Sigma). The medium was
replaced after 24h with DMEM supplemented with 20% FBS
(Invitrogen) and changed every 2 days. On day 6 postinfec-
tion, the cells were trypsinized (trypLE; Invitrogen), passaged
at a 1:6 ratio, and cultured onto one 6-well plate preseeded
with irradiated mouse embryonic fibroblasts (MEF-irr; Glo-
balStem) on a feeder layer of 0.2% gelatin (Sigma). The cells
were grown until spontaneous colony formation using hu-
man embryonic stem cell (hESC) culture media containing
knockout DMEM (Invitrogen) with 20% knockout serum
(Invitrogen), 1mM of l-glutamine (Invitrogen), 0.1mM mer-
captoethanol (Millipore), 1% nonessential amino-acid solu-
tion (Invitrogen), and 10ng/mL of bFGF (R&D Systems).
Alkaline phosphatase and immunofluorescent
Alkaline phosphatase staining was performed using the
Leukocyte Alkaline Phosphatase kit (Sigma). For immu-
nofluorescent staining, cells were fixed with phosphate-buff-
ered saline (PBS) containing 4% paraformaldehyde (Sigma)
for 10min at room temperature. After washing with PBS, the
cells were permeated using 0.25% Triton X-100 (Sigma) for
10min, then blocked with 10% goat serum (Sigma) for 1h at
room temperature. Finally, the primary antibodies were in-
cubated for 45min at room temperature. The antibodies used
in this study included SSEA3, SSEA4, Tra-1–60, Tra-1-81,
CD117, CD90, CD34, CD45, HLA-DR, HLA-G (Biolegend),
and CD59 (Millipore). The cell nucleus was stained with 1mg/
mL Hoechst 33342 (Invitrogen). In order to immunostain the
contractile cardiomyocytes, cardiac troponin T (cTNT; Ab-
cam), Connexin43 (Sigma), and a-sarcomeric actin (Abcam)
were utilized. To quantify the c-kit (+) MiPSCs, a flow cy-
tometry assay was carried out using a BD LSR analyzer (BD
Bioscience). The antibody used in this study was anti-CD117
(Biolegend). The data were analyzed by Flowjo (Tree Star).
Teratoma formation from MiPSCs
About 2·106MiPSCs were suspended in 100mL PBS with
20% Matrigel (BD Bioscience) and transplanted into the hind
limbs of immunodeficient (SCID) mice. Five weeks after the
injection, tumor formation was observed. Hematoxylin and
eosin (H&E) histological stain was performed to determine
the tissue formation from 3 germ lines.
Cell transplantation and survival in SVJ mice
About 2·106reporter gene (luciferase) transduced mouse
ESCs (mESCs) (E14 cell line), MiPSCs, and hAMSCs were
suspended in 100mL PBS with 20% Matrigel (BD Bioscience)
that were transplanted into the hind limbs of SVJ mice.
Under general anesthesia, concurrent BLI was performed
using the charged-coupled device camera (IVIS spectrum;
Caliper) after an IP injection of D-luciferin at 375mg/kg
body weight. Bioluminescence images were acquired for
30min at a 3min interval using Living image 2.5 (Caliper).
HUMAN AMNIOTIC MESENCHYMAL STEM CELL-DERIVED IPSCS
Reverse transcription–polymerase chain reaction
Total RNA was extracted using Trizol reagent (Invitrogen)
according to the manufacturer’s recommendations. Two mi-
crograms of total RNA was transcribed into cDNA using
Superscript first-strand synthesis system (Invitrogen). The
PCR products were size fractionated by 2% agarose gel
electrophoresis (Invitrogen). For real-time PCR, genes were
amplified using iQ SYBR Green Supermix (Applied Biosys-
tems) and StepOne Plus Real-Time PCR Detection System
(Applied Biosystems). All genes were amplified for 40 cycles.
Specific gene expression was first normalized to GAPDH and
then compared with the control groups. Primer sequences are
shown in Supplementary Table S1 (Supplementary Data are
available online at www.liebertpub.com/scd).
Cardiomyocytes differentiation of MiPSCs
MiPSCs were differentiated toward cardiac lineage fol-
lowing a modified protocol reported by Burridge et al. .
Briefly, MiPSCs were dissociated using TryLE Express (In-
vitrogen), and maintained/adapted onto a 1:400 geltrex (In-
vitrogen)-coated culture surface. Single cells of MiPSCs were
passaged strictly every 3 days. The cells from passage 10 on
geltrex were used for cardiac differentiation. On the day of
study, MiPSCs were allowed to aggregate to form embryoid
bodies (EBs) in a V-shaped bottom 96-well plate with 25ng/
mL bone morphogenetic protein 4 (BMP4; R&D Systems) and
5ng/mL bFGF in a RPMI-1640-based serum-free induction
media for 2 days, followed by another 2 days of culture in
10% FBS in ultra-low cluster plates. On day 4, EBs were ad-
hered on a Matrigel (BD Bioscience)-coated plate and fed with
1% FBS in RPMI-1640-based media. The EBs were then
maintained in this medium, which was changed every 3 days.
On observation of spontaneous contractility of the EBs, beat-
ing cells were characterized by flow cytometry, immunostain,
L-type calcium2+current imaging, and atomic force micros-
copy (AFM) to measure the force exerted with each beat.
Flow cytometry for beating MiPSCs
Beating MiPSCs were mechanically detached from the
culture surface and dissociated using TryLE Express (In-
vitrogen) for 5min. The cells were fixed and permeabilized
with PharMingen Perm/Fix solution for 30min at 4?C, and
incubated with primary antibody against human cTNT
(1:200; Abcam) overnight at 4?C. The next day, the cells were
stained with fit-C conjugated secondary antibody (1:500;
Abcam) for 30min at room temperature. The expression of
markers was determined by FACS Calibur (BD Bioscience)
and FlowJo software (Tree Star) to quantify the percentage of
cTNT+ cells. The cells were stained with mouse IgM isotype
antibodies (Biolegend) that were used as the control group.
Live-cell calcium imaging for the contractile MiPSCs
The contractile EBs were mechanically detached from the
tissue culture surface and dissociated with TrypLE Express
for 5min. Single cells were allowed to attach onto gelatinized
glass coverslips (Lab-Tek; Thermo Scientific/Nunc) for 5
days before imaging. On the day of imaging, the cells were
stained with 5mM Fluo-4 AM and 0.02% Pluronic F-127
(Invitrogen) for 15min, and a fluorescence signal from in-
tracellular Ca2+was monitored using fast-line scanning
(1.92ms/line) on a confocal microscope (LSM 510 Meta; Carl
Zeiss) with a ·63 lens (NA=1.4) . Images were further
analyzed using ImageJ software.
AFM for beating MiPSCs
Contractile MiPSC-EBs were mechanically detached from
the tissue culture surface and dissociated with TrypLE Ex-
press for 5min. Single MiPSC were allowed to attach onto
glass bottom plates for 3 days before the imaging. The cul-
ture media of MiPSCs were changed to a media containing
89% Tyrode’s buffer, 10% fetal bovine serum (FBS), and 1%
antibiotic-antimycotic (Cellgro) before the experiment. Cells
were maintained at 36?C during the measurement. The
beating of cells was measured by AFM (MFP-3D Bio) using a
silicon nitride cantilever (spring constants *0.1N/m; PPP-
ContAu, NanoSensors). Cells were gently contacted by the
cantilever tip with 400 pN of force. The cantilever tip re-
mained in the position without Z-piezo feedback, and the
deflection data were collected at a sample rate of 1 kHz for
2min. The deflection trajectory was converted to force tra-
jectory by multiplying by the spring constant and analyzed
by using a Matlab (Mathworks) program. In the force tra-
jectory, the amplitude of beating peaks provided the mea-
surement of cell beating force. The full width at half
maximum of beating peaks measured the beat duration. The
reciprocal of the peak-to-peak interval measured the beating
Leukocyte-mediated cytotoxicity assays
In order to examine the immune characteristics of MiPSCs,
a leukocyte-mediated cytotoxicity assay was performed. One
week after an intramuscular injection of MiPSCs, hESCs (H7
line; Wicell), MEFs (derived from syngeneic SVJ strain), mouse
iPSCs (derived from syngeneic SVJ strain), and hAMSCs into 2
SVJ mice per cell type, the mice were scarified for splenocyte
isolation. The spleen was placed into a 70Um cell strainer (BD
Biosciences) that was preplaced in a Petri dish with cold 20mL
RPMI-1640 (Invitrogen). Using the plunger end of the syringe,
the spleen was forced through the cell strainer into the Petri
dish. The cell strainer was rinsed with 5mL RPMI-1640 and
discarded. The suspended cells were transferred to a 15mL
conical tube and centrifuged at 1,200rpm for 5min at lower
temperature (4?C–8?C). The supernatant was discarded and
resuspended in a pellet in 1mL ACK lysis buffer (Invitrogen).
The cells were incubated at room temperature for 5–10min,
and 9mL of RPMI-1640 were added and spun as earlier. The
cells were resuspended in 5mL RPMI-1640 with 10% FBS and
1% penicillin and streptomycin (PS). The cells were transferred
into a 25cm2flask, incubated at 37?C, 5% CO2, and 95% hu-
midity for at least 2h to remove adherent cells. After 2h, the
splenocytes were cocultured with MiPSCs, MEF, H7, mouse
iPSCs, and c-kit (+) hAMSCs in 24-well plates. After 3 days,
the cytotoxic effects were measured by glucose-6-phosphate
dehydrogenase (G6PD) released from damaged cells using the
Vybrant Cyto-toxicity Assay Kit (Invitrogen).
Data were presented as the mean–standard deviation.
A 2-way analysis of variance was performed. The Tukey–
2800 GE ET AL.
Kramer post hoc test was used for all pair-wise comparisons,
and statistical significance was set at P<0.05. All statistical
analyses were performed using the JMP statistical software
package (SAS Institute).
Immunomodulatory properties of hAMSCs
The hAMSCs were isolated from the amniotic membrane
of the placenta. This population of hAMSCs demonstrated a
high expression of CD59 by immunohistology, presence of
HLA-G, and absence of HLA-DR by reverse transcription–
polymerase chain reaction (RT-PCR) assays (Fig. 1A, B).
Human HLA-G is overexpressed in the placenta to protect
the fetal allograft during pregnancy . In the human
MSCs, HLA-G contributes to the immunomodulatory prop-
erties that allow MSCs to block alloimmune reactions [17,18].
Soluble HLA-G (sHLA-G) molecules produced by the pla-
centa induce the apoptosis of activated CD8+ T-cells and
inhibit CD4+ T-cell proliferation, which play a significant
role in the immunosuppressive effects of the hAMSCs. CD59,
a complement regulatory protein, prevents complement-
mediated cell damage through the inhibition of the com-
plement-mediated membrane attack complex .
Robust generation of iPSCs from hAMSCs
(MiPSCs) and teratoma formation
The characteristic colony formation resembling hESC col-
onies was observed around day 10 (Fig. 2A). The MiPSCs
rapidly expanded on MEF feeder cells using hESC media. In
this study, *200Oct4 positive colonies were found from
50,000 cells on day 18. However, from the fibroblast-derived
iPSCs, 150 Oct4 positive colonies were found from 500,000
cells on day 34. These findings resulted in reprogramming
the frequency of hAMSCs on day 18 to be 0.4% (200/50,000),
while the fibroblasts on day 34 exhibited 0.03% (150/500,000)
reprogramming frequency (Supplementary Fig. S1).These
findings were confirmed by flow cytometry and real-time
PCR, similarly demonstrating the high reprogramming effi-
cacy of hAMSCs on day 14. Flow cytometry revealed *8-
fold higher Oct4 positive cells, while RT-PCR demonstrated
20-fold higher Oct4 expression (Supplementary Figs. S2 and
S3). Morphologically, the characteristic iPSC colonies were
observed on day 20, which displayed a cobblestone ap-
pearance with prominent nucleoli and distinct individual cell
border associated with high activity of alkaline phosphatases
similar to the hESCs but not found in the hAMSCs (Fig. 2B).
2·106MiPSCs were transplanted into the hind limbs of
immunodeficient (SCID) mice. Five weeks after the injection,
tumor formation was observed. H&E histological stain
demonstrated evidence of 3 germ lines (Fig. 2C): epidermis
(ectoderm), muscle (mesoderm), and gut-like epithelium
MiPSCs displayed typical features of H7 hESCs
The hAMSCs demonstrate the partially pluripotent prop-
erties by the expression of SSEA3, SSEA4, and TRA-1-81(Fig.
3A). This unique characteristic of the hAMSCs has proved to
be beneficial for iPSC generation. The RT-PCR data showed
that MiPSCs expressed many hESC-pluripotency marker
genes, including Oct4, Sox2, Nanog, growth, and differenti-
ation factors 3 (GDF3), fibroblast growth factor 4 (FGF4), and
Rex1 (Fig. 3B).Immunostaining results demonstrated that the
MiPSCs expressed hESC-specific surface antigens, including
strates the specific immune characteristics of the hAMSCs: (+)CD59, (+)HLA-G, and (-)HLA-DR. Hoechst 33258 was used
for nuclei staining. (B) Reverse RT-PCR analysis similarly confirms the (+)CD59, (+)HLA-G, and (-)HLA-DR profile of
hAMSCs. hAMSCs , human amniotic mesenchymal stem cells; RT-PCR, reverse transcription–polymerase chain reaction.
Immunostain and reverse RT-PCR of the immunomodulatory properties of hAMSCs. (A) Immunostain demon-
HUMAN AMNIOTIC MESENCHYMAL STEM CELL-DERIVED IPSCS
SSEA3, SSEA4, Tra-1-60, and Tra-1-81(Fig. 3C). These find-
ings were notable, as Sox2, Rex1, and Tra-1-60 were not seen
in the native population of hAMSCs (Fig. 3D).
MiPSCs retained the immunomodulatory
properties of hAMSC
The MiPSCs expressed high levels of HLA-G and CD59,
while the expression of HLA-DR [major histocompatibility
complex (MHC) class II] and HLA-C (MHC class I) were
not detected by immunohistology and RT-PCR assays (Fig.
4A, B). These findings suggested that the absence of MHC
Class I and II in the MiPSCs will enable transplantation
across the MHC barriers to overcome some of the major
immunologic obstacles faced by these cells. Furthermore,
the positive expression of CD59 and HLA-G and the ab-
sence of HLA-DR and HLA-Cin the MiPSCs indicate that
the immunomodulatory property of the hAMSCs is
retained during the reprogramming process. These char-
acteristics were not observed in the H7 hESCs (Fig. 4A).
Real-time PCR was performed to measure the expression of
HLA-G in MiPSCs, hAMSCs, H7 hESCs, and iPSCs from
fibroblasts. The results revealed that HLA-G and CD59
were only expressed in hAMSCs and MiPSCs. The markers
were found in neither H7 hESCs nor iPSCs from fibroblasts
(Supplementary Fig. S4).To examine the immune charac-
teristics of MiPSCs, 2 immunocompetent SVJ mice under-
went an intramuscular injection with each of the following
cell types: MiPSCs, H7 hESCs, MEFs (nonsyngeneic), mouse
iPSCs (syngeneic), and hAMSCs. One week after the in-
tramuscular injection, the splenocytes of the recipient mice
were removed and cocultured with each cell type. The
leukocyte-mediated cytotoxicity was assessed by measur-
ing the G6PD release in each coculture. The cytotoxicity in
the splenocyte cocultures containing the H7 hESCs and
MEFs significantly increased when compared with the
MiPSCs and hAMSCs (P<0.05, n=5, Fig. 4C). Splenocytes
consist of a variety of immune cell populations including T
and B lymphocytes, dendritic cells, and macrophages,
which have different immune functions. These data confirm
MiPSCs from hAMSCs. (A)
Timeline of the MiPSC gen-
eration from the hAMSCs
demonstrates the first ESC-
like colony found within the
first 11 days after transduc-
tion. At day 20, the charac-
teristic iPSC colonies were
observed, which displayed a
cobblestone appearance with
distinct individual cell bor-
der. (B) The MiPSCs demon-
phosphatases activity similar
to the H7 hESCs but that was
not found in the hAMSCs. (C)
Teratoma derived from the
MiPSCs after transplantation
into the hind limbs of SCID
mouse was found 5 weeks
after the injection. Hematox-
ylin and eosin stain con-
contained the tissue from 3
germ lines, including gut-like
epithelial tissues (endoderm),
muscle (mesoderm), and epi-
stem cells; MiPSC, hAMSC-
derived iPSCs; hESCs, human
embryonic stem cells.
Generation of the
2802 GE ET AL.
the immunosuppressive effects from the absence of HLA-
DR to attenuate the response of the T-helper cells to activate
the killer T cells and macrophages and the presence of
HLA-G and CD59 to induce the apoptosis of activated
CD8+ T-cells, inhibit CD4+ T-cell proliferation, and at-
tenuate complement-mediated cytotoxicity [18,20,21].These
findings provide evidence that the transplanted MiPSCs
may survive in vivo by inhibiting the host immune re-
The in vivo study demonstrated robust BLI survival signal
by the luciferase-transduced
hAMSCs transplanted into the mouse hindlimb at week 1
(Fig. 4D). These results verified that HLA-G and CD59
played an important role in the in vivo survival of the
hAMSCs. However, the luciferase-transduced MiPSCs only
survived 5 days in the SVJ mouse, which is contrary to the in
mESCs (syngeneic) and
vitro data and robust survival of hAMSCs. There may be
other nonimmunological reasons for their poor survival,
which is currently under investigation.
C-kit (+) MiPSCs displayed robust
The c-kit (+) hAMSC sub-population represented a highly
selective sub-population. They were sorted by FACS by ex-
cluding the HLA-DR, CD34, and CD45 (immunity and he-
matopoietic markers) cells and by including the SSEA3,
SSEA4, TRA1-60, TRA1-81, thy-1, and c-kit (stemness and
cardiac markers) cells. C-kit (CD117) is considered an im-
portant surface marker of precardiac mesodermal progenitor
cells [22,23]. This marker was not found in the H7 hESCs but
was expressed robustly in the human MiPSCs (Fig. 5A, B).
(+)SSEA3, (+)SSEA4, and (-)Tra-1-60 demonstrating partial pluripotency of the hAMSCs. (B) RT-PCR was performed to
compare a total of 23 ESC marker expressions between the H7 hESCs and MiPSCs. (C) Immunostain of the MiPSCs was
positive for SSEA3, SSEA4, Tra-1-60, and Tra-1-80. Note Tra-1-60 was negative for the hAMSCs. (D) Comparison of the
stemness properties between the hAMSCs and MiPSCs demonstrated the absence of Sox2 and Rex1 in the hAMSCs.
Stemness properties of the hAMSCs and MiPSCs. (A) Immunostaining of hAMSCs demonstrated(+)Tra-1-81,
HUMAN AMNIOTIC MESENCHYMAL STEM CELL-DERIVED IPSCS
Flow cytometry assay revealed that the proportion of c-kit
positive MiPSCs (MiPSC+s) was *54.6% of the total
MiPSCs. IgM isotype for the MiPSCs served as the control
group, demonstrating negligible signals (Fig. 5C).
In order to investigate the cardiac differentiation potential
of MiPSC+s, the cells underwent cardiac differentiation.
Contractile areas were observed at day 12 after induction
with the modified cardiac differentiation protocol  (Sup-
plementary Movies S1 and S2). By day 28, more than 95% of
the EBs generated from the MiPSCs was found to be spon-
taneously beating. Immunohistology and flow cytometry of
the beating cells showed the robust colocalization of cTnT,
connexin 43, and a-sarcomeric actin in the contractile areas
(Fig. 5D). Furthermore, *40% of the cells derived from the
MiPSC+s were found to be (+)cTNT in comparison to the
19.9%* (+)cTnT cells from the unselected MiPSCs, and
4.51%* (+)cTnT cells from H7 cells (*P<0.05, n=3, Fig.
5E).The contractile MiPSC-derived cardiomyocytes exhibited
MiPSCs. (A) Immunostain confirms
that both CD 59 and HLA-G were
positive and HLA-DR was negative in
the MiPSCs (left). However, this pro-
file was not seen with the H7 hESCs
(right). (B) RT-PCR analysis was per-
formed to confirm the absence of
HLA-DR and HLA-C in the MiPSCs.
(C) Splenocytes cocultured with MEF,
hESCs, MiPSCs, mouse iPSCs, and
hAMSCs for 24h. Significantly de-
creased cytotoxicity (P<0.05, n=5)
was observed in the cocultures con-
taining the MiPSCs, hAMSCs, and
mouse iPSCs (syngeneic). (D) Mouse
ESCs, MiPSCs, and hAMSCs were in-
jected into the hind limbs of immu-
nocompetent SVJ mice. The mESCs
and hAMSCs demonstrated robust
BLI survival signals, while the MiPSCs
did not do so at week 1. MEF, mouse
Immune characteristics of the
2804 GE ET AL.
profile of the MiPSCs. (A) C-kit
(CD 117) surface marker was
positive in MiPSCs. (B) C-kit (CD
117) surface marker was negative
in H7 hESCs. (C) Flow cytometry
for MiPSCs demonstrated signif-
icant c-kit (+) MiPSC sub-popu-
lation (arrow). The proportion of
c-kit (+) MiPSCs was *55%. The
control group, IgM isotype stain,
exhibited negligible c-kit expres-
sion. (D) The contractile areas of
the differentiated MiPSCs were
characterized by the colocaliza-
tion of cTNT, Connexin 43, and
alpha-sarcomeric actin. Hoechst
33258 was used for nuclei staining
(blue). (E) Flow cytometry analysis
revealed *40.3% cTNT+
derived from c-kit (+) MiPSCs,
19.9%* cTnT+ cells derived from
unselected MiPSCs, and 4.5%*
cTNT+ cells derived from H7
hESCs (*P<0.05, n=3). The cells
derived from both MiPSCs and
H7 cells were stained with mouse
IgM isotype antibodies to be used
as the control group, respectively.
cTNT, cardiac troponin T.
HUMAN AMNIOTIC MESENCHYMAL STEM CELL-DERIVED IPSCS
the characteristic L-type Ca2+current/transient peaks across
the membrane with each depolarization, as indicated by the
calcium-dependent intracellular fluorescence intensity de-
tected by confocal microscopy (Fig. 6A). Finally, AFM anal-
ysis of the 4 representative contractile MiPSC-derived
cardiomyocytes showed that the maximal contractile force of
each beat was comparable to that of native cardiomyocytes
(10-0.4*10-0.6 nN) (Fig. 6B).In comparison to other pub-
lished studies on the cardiac differentiation of iPSCs,
MiPSC+s demonstrated a robust cardiac differentiation po-
tential and generated an enriched pool of mature cardiac
In this study, the high reprogramming efficiency of the
(MiPSCs) was demonstrated. The MiPSCs retained the im-
munomodulatory and precardiac mesodermal properties of
the hAMSCs. The partially pluripotent property of hAMSCs
marked with an endogenous expression of Oct4 and Nanog
conferred high reprogramming capability. The data revealed
that the ESC-like colony morphology appeared within 10
days, which represents a rapid and expeditious reprogram-
ming process [1,25]. Published reports suggest a 4-week
duration for human iPSC generation from skin fibroblasts,
which could be shortened to *14 to 20 days when enhanced
with small molecules . Some investigations have reported
that the endogenous expression of reprogramming genes by
adult somatic cells facilitates the generation of iPSCs [26,27].
However, an optimal adult somatic target cell population
has not been identified. Although the 4Yamanaka factors
were employed to transduce the hAMSCs into iPSCs, it is
possible that 1 or more Yamanaka factors could be replaced
by the endogenous pluripotency genes. Many studies have
demonstrated that reduced number of Yamanaka factors
such as Oct4 along with either c-Myc or Klf4 can reprogram
adult mouse neural stem cells that endogenously expressed
Sox2 . The hAMSCs exhibit a high expression of Nanog
and Oct4 with positive stem cell surface markers including
SSEA3, SSEA4, Tra-1-81, and thy-1. However, the precise
role of endogenous expression of Nanog and Oct4 in the
rapid reprogramming process is not clear. The current study
suggests that this unique population of hAMSCs represents a
potentially promising source of cells for the robust and rapid
generation of iPSCs that may overcome some critical issues
in iPSC generation.
The MiPSCs maintain an immunomodulatory capability
through the presence of CD59 and HLA-G and the absence
of HLA-DR and HLA-C expression. The primary function of
HLA-DR is to present peptide antigens, potentially foreign in
origin, to the immune system for the purpose of eliciting T-
helper cell responses that eventually lead to the production
of antibodies against the same peptide antigen. On the other
hand, HLA-G plays a role in immune tolerance during
pregnancy for the placenta situated along the maternal-fetal
the contractile MiPSC-derived cardiac cells exhibited a characteristic calcium transient with each depolarization. The left image
represents a time-lapsed fluorescence image of a line scan of confocal stain, and the right image indicates the calcium peaks.
The MiPSC-derived cardiac cells demonstrated mature cardiomyocyte characteristics. (B) The histogram of atomic force
microscopy that evaluates the force exerted by each contraction of a representative MiPSC-derived cardiac cell (left image).
The maximal contractile force measured for 4 representative beating cells was comparable to that of native cardiomyocytes
(right image, 10-0.4*10-0.6 nN).
Electrophysiological characterization of the contractile MiPSC-derived cardiac cells. (A) Live-cell calcium imaging of
2806GE ET AL.
border. The presence of sHLA-G has been associated with
higher pregnancy rates . The sHLA-G molecules induce
apoptosis of the activated T-cells and inhibit CD4+ T-cell
proliferation, effectively inhibiting alloimmune reactions
[18,21]. In addition, CD59, a complement regulatory protein,
prevents complement-mediated cell damage through inhi-
bition of the complement membrane attack complex .
Immunologic reactions are considered a critical issue in
stem cell-based therapy when using nonmatched stem cells.
To address this issue, autologous iPSCs derived from the
patient’s somatic cells are considered a potentially landmark
solution. Even in a situation where an immediate treatment
is unnecessary, a therapeutic strategy to wait for ex vivo
reprogramming and expansion with the subsequent re-
transplantation of autologous stem cells may not be finan-
cially and clinically feasible . Recently, Zhao et al. re-
ported that the cells differentiated from iPSCs can induce T-
cell-dependent immune responses in syngeneic recipients
. This represents a major challenge for iPSC transplan-
tation therapy. Rather, an off-the-shelf supply of universal
iPSC-derived cells may be more preferable. The data from
this study suggest that the MiPSC-derived cells could make
allogeneic iPSC transplantation therapy possible.
Finally, the MiPSCs retain the c-kit (CD117) cell surface
marker (MiPSCs+). As described in many studies, c-kit is an
important surface marker of cardiac progenitor cells in adult
stem cells and has not been found in ESCs . In 2009,
Okabe and colleagues reprogrammed c-kit (+) hematopoi-
etic cells from mouse to pluripotent stem cells by the direct
viral transfer of iPSCs factors, but the c-kit expression did not
persist in the iPSCs . In our current study, the MiPSC+s
derived from the hAMSCs demonstrated enhanced cardiac
differentiation efficiency through a high percentage of
spontaneous contractility in a short period of time. Further-
more, these MiPSC+-derived cardiac cells exhibited consis-
tent development into a mature cardiomyocyte phenotype.
Cardiac differentiation from iPSCs holds great promise for
regenerative medicine; however, low reprogramming effi-
ciency and suboptimal differentiation efficiency limit cardi-
omyocyte generation. Various molecular strategies, small
molecules, and peptides led to numerous cardiac differenti-
ation protocols [24,32–34]. However, the efficacy of these
approaches was modest. An alternative solution is to select a
cell population that may be predisposed to high repro-
gramming and cardiac differentiation efficiency.
Our study demonstrated that the unique characteristics of
MiPSCs+may enable a robust, rapid, and safe method for
generating universal, off-the-shelf supply of cardiac lineage
committed cells. The findings validated a cell-specific ap-
proach to translate novel stem cell biology to induce plur-
ipotency and cardiac differentiation.
This study is funded by NIH R01 HL097516 (PY). The
authors thank Dr. Xiaoxing Xiong and Dr. Peng Wang for
their technical assistance in the cell imaging.
Author Disclosure Statement
Boehringer-Ingelheim: research support; General Electric
Healthcare: research support.
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Address correspondence to:
Dr. Phillip C. Yang
Division of Cardiovascular Medicine
Department of Medicine
School of Medicine
300 Pasteur Drive, H-2157
Stanford, CA 94305
Received for publication August 4, 2011
Accepted after revision April 24, 2012
Prepublished on Liebert Instant Online April 24, 2012
2808 GE ET AL.