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0007 -4888/13/15540552 © 2013 Springer Science+Business Media New York
Activation by ACA Induces Pluripotency
in Human Blood Progenitor Cells
Z. A. Becker-Kojić, J. R. Ureña-Peralta, I. Zipančić,
F. J. Rodriguez-Jiménez, M. P. Rubio, P. Stojković,
M. G. Roselló, and M. Stojković
Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 2, pp. 85-101, April, 2013
Original article submitted August 12, 2012
Reprogramming of human somatic cells by transcription factors to pluripotent state holds
great promise for regenerative medicine. However, low effi ciencies of current reprogramming
methods, immunogenicity and lack of understanding regarding the molecular mechanisms
responsible for their generation, limits their utilization and raises questions regarding safety
for therapeutic application. Here we report that ACA signaling via PI3K/Akt/mTor induces
sustained de-differentiation of human blood progenitor cells leading to generation of ACA
pluripotent stem cells. Blood-derived pluripotent stem cells differentiate in vitro into cell
types of all three germ layers, exhibiting neuronal, liver, or endothelial characteristics. Our
results reveal insight into the molecular events regulating cellular reprogramming and also
indicate that pluripotency might be controlled in vivo through binding of a natural ligand(s)
to ACA receptor enabling reprogramming through defi ned pathway(s) and providing a safe
and effi cient method for generation of pluripotent stem cells which could be a breakthrough
in human therapeutics.
Key Word: ACA glycoprotein; human oocytes; embryonic stem cells; pluripotency; dif-
ferentiation
Principe Felipe Research Centre Valencia, Spain. Address for cor-
respondence: becker-kojic@gmx.de. Z. A. Becker-Kojić
Human embryonic stem cells (hESCs) are character-
ized by two distinctive properties, their pluripotency
e.g., ability to differentiate into all derivatives of three
primary germ layers and their capability to self-renew
themselves indefi nitely, thus making them interesting
for regenerative medicine and tissue replacement after
injury or disease. Besides the ethical concerns of em-
bryonic stem cell therapy, there is the immunological
problem of graft-versus-host disease associated with
allogeneic stem cell transplantation. This problem is
now to be solved through induced pluripotent stem
cells (iPSCs) which are the product of somatic cell
reprogramming to an embryonic-like state [18,24].
The original protocol is based on retroviral mediated
introduction of various transcription factors Oct4,
Sox2, Klf4, and c-Myc (OSKC) or through other factor
combinations which are able to induce pluripotency.
Although the major obstacle for safe personal-
ized stem cell therapy has been overcome by using
non-integrating vectors like piggyBac technology [22],
adenoviruses, plasmids [10,14], or miRNA [2] in or-
der to achieve transient expression of reprogramming
factors, a main issue regarding safety of iPSCs still
exists. Despite great progress, the cell origins and un-
derlying molecular mechanisms, by which somatic
cell nuclei are reprogrammed to pluripotency, remain
elusive [12].
Many factors which were initially supposed to
be essential for iPSC induction and generation in the
meantime proved to be non-essential or even irrel-
evant. Reprogramming is slow, ineffi cient and a sto-
Cell Technologies in Biology and Medicine, No. 2, August, 2013
553
chastic process [9] and often incomplete. Only 0.01-
0.02% of somatic cells undergoes reprogramming by
these methods, suggesting the existence of randomness
due to heterogeneous mechanisms occurring through-
out reprogramming, depending on whether the somatic
stem cell or fully differentiated tissue-specifi c cell is
undergoing this process [17]. Although claimed to be
the solution for overcoming immunological problems
related to use of human embryo for the derivation
of pluripotent stem cells it was proven recently, that
iPSCs cause host T cell-dependent immunological re-
sponse to the transplanted tissues in syngeneic animal
in contrast to the cells derived from ESC [26]. Another
unresolved problem is teratoma, an indispensable cri-
terion for pluripotency, because of cancer potentially
arisen from the residual number of undifferentiated
pluripotent stem cells.
Here, we address the question of about the role
of ACA in early human development and studied the
ACA specific signaling mechanism and its poten-
tial for reprogramming human cells to pluripotency.
Moreover, the study of specifi c signaling pathway(s)
regulated by ACA could provide insights into possibly
occurring natural pluripotency in humans.
RESULTS
Expression of ACA in human egg,
embryo, and ESCs
As embryo is the “mother” of all stem cells we sought
to examine ACA expression in human oocyte and pre-
implantation embryos. Fixed human eggs, as well as
1, 2, 3, and 5 days post in vitro fertilized embryos
were stained with monoclonal antibody specifi c for
ACA. We observed massive and sustained expression
of ACA, throughout human embryonic development
starting with the egg (Fig. 1, a-b). All early embryonic
stages from oocyte, through 1-5 cell embryos until the
blastocyst stage, showed robust and massive expres-
sion of ACA (Fig. 1, c-g). Moreover, we found some
clearly distinctive microdomain hot spots with very
intense ACA expression compared to other parts of
human one-cell embryo (Fig. 1, c).
ESCs are pluripotent stem cells derived from the
inner cell mass of the blastocyst in early stage em-
bryo [7] 4-5 days post-fertilization, at that time point
they consisted of 50-150 cells. These cells retain the
developmental potency of foetal founder cells that
undergoes symmetrical self-renewal in culture, appar-
ently without limit. As displayed in Figure 2, a, hESC
line H9 shows massive expression of ACA. In these
cells, ACA is co-expressed with hESC-specifi c surface
antigen, like tumor-related antigen TRA-1-81.
ACA is involved in maintenance of pluripotency
Establishment and maintenance of the pluripotent
state of hESCs is a key issue in stem cell biology
and regenerative medicine; identifi cation of factors
that regulate ESC is an important goal. It is generally
believed that derivation, propagation and maintenance
of pluripotency of ESCs relay on manifold stimulating
factors implemented in vitro by empirical combina-
tions of feeder cells, conditioning media, cytokines,
and growth factors which are part of its signaling net-
work. Pluripotent ESCs can be maintained in an un-
differentiated state in culture using appropriate feeder
cells and growth factors, but they are poised for rapid
differentiation [21].
We examined whether ACA might be involved in
the maintenance of pluripotency by analyzing H9 cells
after they have started to spontaneously differentiate
in culture. The differentiating cells grew no longer
in colonies and the hESC marker got lost upon dif-
ferentiation (Fig. 2, b). To explore whether initiation
of ACA signaling pathway(s) could rescue a pluripo-
tent phenotype of hESCs, we activated ACA at the
surface of spontaneously differentiated H9 cells by
means of crosslinking using ACA specifi c antibody
[3]. The pluripotent phenotype of hESCs was fully re-
stored upon activation by ACA, showing expression of
TRA-1-81 and TRA-1-60 (Fig. 2, c). This experiment
demonstrated that activation by ACA maintains the
undifferentiated state of hESCs strongly implicating
its involvement in signaling network which is critical
for maintenance of the ground state of pluripotency.
Turning the clock back
We next experimentally tested whether ACA signaling
network, once initiated, could promote reprogram-
ming of human blood progenitor cells to pluripoten-
cy. Mononuclear cells isolated from peripheral blood
(PBMNCs) using a standard protocol with Ficoll were
treated with ACA specifi c antibody, as previously de-
scribed, and propagated for 16 days in Iscove’s media
without addition of growth factors or cytokines.
Time course fl ow cytometry analysis was used
to investigate the modifying phenotype of de novo
generated ACA stem cells during the culture time pe-
riod. Over the course of 16 days we observed a pro-
gressive decrease of markers characteristic for adult
progenitor cells with more differentiated phenotype
like HLA-DR, CD38 and CD34 in growing popula-
tion, and substantial increase of marker characterizing
very primitive phenotype like receptor tyrosine kinase
(CD117), also known as c-Kit. The population of cells
with more primitive phenotype, baring a c-Kit marker,
considerably increased and two weeks later virtually
no HLA-DR, CD38, or CD34 positive cells were pres-
ent in the new ACA-generated stem cell population
(Fig. 3, a).
Z. A. Becker-Kojić, J. R. Ureña-Peralta, et al.
554
As assessed by fl ow cytometry, continued cul-
turing of ACA-activated PBMNCs in Iscove’s media
for 16 days triggered the process of de-differentiation
and rendered the great majority of these cells plu-
ripotent. ACA-generated cells showed co-expression
of ES cell-specifi c markers like extracellular matrix
sialylated keratin sulfate proteoglycan (SSEA-4) and
tumor rejection antigen TRA-1-61 (Fig. 3, b). The
same experiment was performed with various donors
and a summary of results is presented in Figure 3, c.
In order to analyze the expression of genes char-
acteristic of undifferentiated hESC we performed
semi-quantitative RT-PCR analysis at different
stages during culture time period of ACA activated
PBMNCs. NANOG transcript appeared to be the fi rst
at day 5, its expression gradually increased during
culture time, followed by SOX2 and TERT at day
8, and REX1 at day 12. The level of transcripts was
similar to that of hES line H9 (Fig. 3, d). Confocal
analysis revealed that size and morphology of these
cells changes during the time indicating a reprogram-
ming effect that occurred along the way of culturing
cells upon activation by ACA. The initial cell sizes
decreased up to 10 times, the cells grew in colonies,
were very small, with 1-2 nm in diameter, they dis-
played several features typical for ESCs, such as a
large nucleus surrounded by narrow rim of cytoplasm
(Fig. 3, e).
On day 12, after the activation of the PBNCs cul-
tures by ACA, the majority of reprogrammed cells
made up the fi rst small colonies which express ESC
markers, SSEA-4, TRA-1-61 and TRA-1-81 (Fig. 3, f)
whilst just a few cells still expressed SSEA-1 antigen,
a marker characteristic for differentiated hESCs and
therefore not expressed on pluripotent stem cells. This
fi nding suggests that the route to pluripotency in ACA-
specifi c manner is exactly opposite to that known for
differentiation of hESCs. Surrounded cells represent
Fig. 1. Oocyte and embryonic expression of ACA at the designated age.
Immunophenotyping of fixed human egg and embryo with antibody specific to ACA
revealed expression of a novel GPI-linked surface glycoprotein ACA in these cells.
DAPI was used to visualize DNA. Micrographs are taken by confocal microscopy.
a) Human eggs, (negative control).
b) Human egg stained with ACA-specific antibody and mount with DAPI.
c-g) In vitro post fertilized human embryo on days 1 -5, stained with ACA-specific
antibody versus DAPI.
Cell Technologies in Biology and Medicine, No. 2, August, 2013
555
terminally differentiated blood cells, which cannot be
reprogrammed by ACA method.
ACA signaling pathway(s)
As shown previously (Becker-Kojić et al., submitted),
ACA regulates a mechanism which determines the fate
of human HSCs initiating via PI3K/Akt/PTEN/mTor
a large and complex signaling machinery, which up-
regulates c-Kit, Notch and Wnt pathways known to
play a role during the course of human development.
To look closer at the mechanisms whereby ACA
initiates a de-differentiation process, which leads to in-
duction of pluripotency, we estimated ACA-mediated
phosphorylation as an important regulatory mecha-
nism in these cells. Protein extracts of PBMNCs be-
fore and 6 days after activation by ACA are subjected
to western blot analysis with various phospho-specifi c
antibodies to analyze the expression and phosphoryla-
tion status of proteins potentially implicated in ACA-
specifi c signaling pathway(s).
Our results demonstrate that ACA activation phos-
phorylates, and thereby induces, signals via PI3K/Akt
(Fig. 3, g). Considering ACA protein as a membrane
receptor, we investigated in-depth the specifi c proteins
whose activity is known to modify as a result of recep-
tor occupancy by its natural ligand(s). Obvious candi-
dates for this type of cell activation are the enzymes
involved in pathways affected by the growth factor
binding and receptor kinase activation.
Phospatidylinositol phospholipase C (PLC) is
a family of cellular proteins, which plays a central
role in signal transduction pathways. Certain growth
factors appear to stimulate cellular PLC activity by
receptor-mediated tyrosine phosphorylation of PLCγ
1 isozyme. As expected, we found that PLCγ1 and
Fig. 2. Expression of ACA in hESC line H9.
hESCs derived from H9 cell line grown on feeder cells were fixed and stained with antibody specific for ACA and hESCs marker. Micro-
graphs are taken by confocal microscopy.
a) A colony of hESCs stained with ACA and TRA-1-81 antibodies.
b) Spontaneously differentiated ESCs stained with TRA-1-81 antibody. DAPI staining indicates the total cell content per field.
c) ACA activated, spontaneously differentiated ESCs stained with TRA-1-81antibody. Staining with DAPI indicates the cell number per field.
Z. A. Becker-Kojić, J. R. Ureña-Peralta, et al.
556
Fig. 3. Generation and analysis of blood derived pluripotent stem cells.
MNCs isolated from steady-state peripheral blood were crosslinked with ACA-specific antibody followed by Alexa 488 labeled goat anti-
mouse and phenotype of new generated cells analyzed by means of fluorescence cytometry, semi-quantitative RT-PCR analysis, western
blotting and confocal imaging.
a) A representative flow cytometry histogram showing de-differentiation of more mature blood progenitor cells in PBMNCs and generation
of primitive ACA/c-Kit population not present in steady-state PBMNCs.
b) FACS analysis of hESCs markers SSEA-4 and TRA-1-81 in pluripotent stem cells generated upon activation by ACA.
c) PBMNCs obtained from various donors activated by means of ACA crosslink leads to generation of HSCs in a week one, and pluripotent
stem cells in a week two of culture time period. 1) CD34; 2) TRA-80-1; 3) SSEA-4. Data are averaged and represented as mean±SEM.
Cell Technologies in Biology and Medicine, No. 2, August, 2013
557
PLCγ2 are phosphorylated upon activation by ACA.
Of the multiple isoforms of PI3 kinases, the class I
PI3K that comprise α, β, and γ p110 catalytic subunits
as well as p85 adapter regulatory subunit, which re-
cruits the catalytic subunits to the plasma membrane is
phosphorylated/activated in an ACA-dependent man-
ner. Phosphorylation of 3-Phosphoinositide–dependent
kinase 1 (PDK1), the major transducer of PI-3-kinase
action and responsible for upstream activation loop
of Akt which is central to promote cell survival by
inhibiting apoptosis, is also activated in ACA-specifi c
manner. As expected, ACA/PI3K/Akt down-stream
Fig. 3. Generation and analysis of blood derived pluripotent stem cells (continued).
d) RT-PCR analysis of the ES marker genes NANOG, REX1, SOX2, and TERT, in ACA-generated pluripotent stem cells.
e) Images of blood-derived mononuclear cells showing morphology changes from single cells on day 1 to small embryonic-like colonies
with large nuclei and scant cytoplasm on day 12, during ACA reprogramming procedure.
f) ACA stem cell colonies express markers common to pluripotent stem cells including SSEA-4, TRA-1-60, and TRA-1-81.
g) Phosphorylation pattern of PBMNCs before and 6 days after ACA activation. The expression of phosphorylated PI3K and Akt as deter-
mined by Western blot analysis were diminished when the cells were grown in the presence of ET. Non-activated mononuclear cells (NA)
isolated from peripheral blood are used as control. A) FCF-fctivated MNCs; ET) inhibitor.
h) RT-PCR analysis of pluripotency marker genes on ACA-generated cells during the culturing in the presence or absence of PLC inhibitor ET.
Z. A. Becker-Kojić, J. R. Ureña-Peralta, et al.
558
signaling also includes up-regulation of glycogen syn-
thase kinase 3 (GSK-3), key regulators of numerous
signaling pathways, as well as a mammalian target
of rapamycin (mTOR) complexes, which are crucial
regulators of translation initiation and protein syn-
thesis [5,23]. The inhibition of PLCγ with ET-18-0-
CH3 (ET) reduces the extent of protein expression and
phosphorylation of PI3K/Akt (Fig. 3, g) indicating that
the ACA signaling cascade is organized in a hierarchi-
cal manner. The blocking of PLCγ greatly reduces the
generation of CD34 cells, which is an inevitable step
in introducing pluripotency in human progenitor cells,
whereas inhibition of PI3K alone with its specifi c in-
hibitor LY294002 (LY) had no effect on the generation
of CD34 cells, thereby confi rming the results obtained
by Western blotting. Most importantly, inhibition of
PLCγ with ET diminished, but together with the in-
hibition of PI3K with LY it virtually abolished the
generation of these cells.
The expression of pluripotency gene markers
NANOG, TERT, OCT3/4 and REX1, induced upon
activation by ACA is greatly reduced or impaired by
treatment with ET, thus confi rming that the activation
of PLCγ via PI3K/Akt signaling mechanism, previ-
ously described for the generation of self-renewing
HSCs, is responsible for induced pluripotency in ACA
stem cells (Fig. 3, h).
ACA-generated stem cells express
hESC-specifi c antigens
Next, we assessed these newly generated pluripotent
stem cells through standard criteria established for
hESCs, such as morphology, marker expression, geno-
type, and in vitro as in in vivo differentiation.
Culturing of PBMNCs in mouse embryonic con-
ditioned media (CM) or Knockout–Dulbecco’s Modi-
fi ed Eagle Medium (DMEM) for 2 weeks, upon ACA
activation, generates cells, which grew and formed
colonies on mouse embryonic feeder cells (MEF), as
previously reported for hESCs (Fig. 4, a). Surpris-
ingly, we found that ACA-generated pluripotent stem
cells grew in large colonies in a suspension just as
well (Fig. 4, b).
We performed RT-PCR on both cell popula-
tions and confi rm ed that ACA reprogrammed cells
expressed hESC marker genes such as endogenous
OCT3/4, SOX2, NANOG, TERT and REX1 on similar
levels or higher than those in hESC line H9, irrespec-
tive of culture conditions (Fig. 4, c). In parallel, the
expression of OCT3/4, NANOG, SOX2 and REX1 was
gradually reduced or abolished when ACA-generated
pluripotent stem cells undergo spontaneous differen-
tiation (Fig. 4, e). Upon activation by ACA, as was
precisely for H9 cells, differentiated cells returned to
their undifferentiated pluripotent state by recovering
the expression of genes required for maintenance of
pluripotency (Fig. 4, d-e).
Our results showed that the derivation and propa-
gation of pluripotent stem cells by ACA are not depen-
dent on environmentally derived signals like specifi c
media or feeder cells, but most likely the intrinsic
factors rather than extrinsic ones regulate the develop-
ment of pluripotency in these cells.
By means of immunochemistry, we analyzed
ACA-generated stem cells for expression of markers
shared with hESCs [20]. ACA pluripotent stem cells
grown on matrigel were organized into colonies. Con-
sistent with morphology and size similar to hESCs,
ACA cells were positive for TRA-1-60, TRA-1-81,
and SSEA-4 (Fig. 5, a-c).
ACA-generated pluripotent stem cells growing
in suspension show the same immunophenotype as
cells growing on matrigel. These cells were positive
for SSEA-4, TRA-1-60 and TRA-1-81 in concor-
dance with hESC (Fig. 5, e-g). Fully reprogrammed
ACA stem cells did not express stage-specifi c em-
bryonic antigen SSEA-1, which is a characteristic
of differentiated ESCs, except for a few cells at the
edge of the colonies (Fig. 5, g). These data confi rmed
that reprogramming by ACA strictly followed de-
differentiation pattern, starting from more mature
phenotype towards the cells with the most primitive
phenotype, e.g. pluripotent stem cells. We found that
ACA-generated pluripotent stem cells do not form
teratoma when transplanted into NOD-SCID mice
(data not shown).
In vitro and in vivo differentiation
of ACA pluripotent stem cells
To assess in vitro differentiation capacity of ACA-
generated pluripotent stem cells using specifi c ex vivo
culture condition that mimic the in vivo microenvi-
ronment of a given tissue, we explored whether ACA
pluripotent stem cells have the developmental poten-
tial to spontaneously differentiate into several somatic
cell type of endodermal, mesodermal, and ectodermal
origin of all three primary germ layers.
We next examined whether ACA cells can be in-
duced to generate the cells having neural characteri-
stics.
The PBMNCs upon activation by ACA were
grown in suspension in Iscove’s media as described,
and 10 days later seeded on fi bronectin-coated culture
plates in neural differentiation media (GRM) supple-
mented with bFGF, a cytokine known to support neu-
ral stem cell (NSC) proliferation according to recently
reported protocol. As early as 5 days after starting
the targeted differentiation of ACA cells, we detected
the fi rst neuronal cells with long branching structure.
Notably, over time from day 5 to day 30 we observed
Cell Technologies in Biology and Medicine, No. 2, August, 2013
559
Fig. 4. Growing of ACA-generated pluripotent stem cells.
ACA-generated pluripotent stem cells were grown in suspension or on mouse feeder cells. Semi-quantitative RT-PCR assay was used for
expression analyses of ESCs marker genes in ACA-generated pluripotent stem cells.
a) Morphology of ACA-generated pluripotent stem cells growing on MEF layer in CM. The micrographs were taken using phase contrast
microscopy.
b) Morphology of ACA-generated pluripotent stem cell colonies floating in liquid culture in Iscove’s medium. Images were obtained by
confocal microscopy.
c) RT-PCR of ES marker genes in PBMNCs, H9, and ACA-generated pluripotent stem cells grown in suspension in Iscove’s medium, or
on feeder cells in CM.
d) Phase contrast image of spontaneously differentiated colony of ACA-generated pluripotent stem cells before and after ACA treatment.
I) spontaneous differentiation of ACA cells; II) ACA activation.
e) RT-PCR analysis of pluripotent marker genes in spontaneously differentiated ACA stem cells colonies before and after activation by ACA.
Z. A. Becker-Kojić, J. R. Ureña-Peralta, et al.
560
increasingly complex morphologies characteristic for
neuron progenitors, oligodendrocytes and astrocytes,
suggesting an active process of neuronal maturation in
newly formed cells (Fig. 6, a).
We assessed neuronal differentiation of ACA cells
by means of a semi-quantitative RT-PCR analysis.
These cells express various markers like neural cell
adhesion molecule (NCAM), which is a homophilic
binding glycoprotein expressed on the surface of neu-
rons and glia. Moreover, ACA-generated cells with
neuroectodermal characteristics express neuron-spe-
cifi c class III β-tubulin Tuj1 which is a marker abun-
dantly found in brain, where it is expressed almost
exclusively in neurons after differentiation, as well
as nestin, an intermediate fi lament protein expressed
mostly in nerve cells, where it is implicated in radial
growth of the axon. RT-PCR analysis featured a panel
of many other markers, characterizing various types
and stages of neuroectodermal differentiation (Fig. 6,
d). These neuronal markers were not present in de-dif-
ferentiated ACA cells generated after ACA activation,
or in starting population consisting of mononuclear
cells isolated from peripheral blood, but only in the
cells that underwent targeted neural differentiation
after growing in defi ned media. Immunocytochemistry
confi rms the expression of Tuj1 and nestin in neuronal
ACA cells as well as O4, an early oligodendrocyte
marker. These data confi rm that blood-derived ACA
stem cells possess a potential to differentiate into ec-
toderm represented by neuroectodermal cells and indi-
cates a potential of these cells to turn from blood cells
into brain cells.
Next, we investigated whether ACA pluripotent
stem cells can be differentiated into endodermal lin-
eages. To address this question we cultured ACA-
generated cells according to two different original
primary culture systems. One was a reported method
with a combination of growth/differentiation factors,
like leukemia inhibitory factor (LIF), a recombinant
human stem cell factor (hSCF), recombinant human
oncostatin M (OSM) as well as a recombinant human
hepatocyte growth factor (hHGF) which is used for
endodermal differentiation. We also developed our
own protocol using solely hHGF and compared the
effi ciency for hepatic differentiation of these two sys-
tems. To investigate whether the cultured round cells
derived from ACA-generated stem cells produce he-
patocyte lineages, we examined the expression pattern
of differentiation markers for endodermal lineages.
The total RNA was extracted from ACA cells at vari-
ous stages of differentiation and semi-quantitative RT-
PCR performed to examine the expression profi le of
newly generated cells. The analysis revealed that the
transcripts of human glycogen synthase (GS), known
to be expressed in mature hepatocytes, cytokeratin 18
(CK-18), characteristic for hepatocyte lineages, and
α-fetoprotein (AFP) expressed in hepatic progenitor
cells, appeared at day 7 and strongly increased at day
18. Cytokeratin 18 transcript was similar during the
culture time period, whereas CK19, which is a marker
of hepatic progenitor cells, was initially present at
day 7 and down-regulated after 2 weeks indicating
the presence of mature hepatocytes in culture. Above
all, ACA-generated cells displayed the characteristics
of functionally differentiated hepatocytes, expressing
releasing human albumin (ALB), which increases sub-
stantially in the process of maturation (Fig. 6, e). Im-
munofl uorescence analysis revealed that the cultured
cells express AFP and ALB, thus confi rming endoder-
mal differentiation of ACA reprogrammed cells (Fig.
6, b). Taken together, our data demonstrate that ACA
cells can differentiate into cells of various hepatocyte
lineages including both, hepatic progenitor cells and
mature hepatocytes. Most importantly, only the signals
triggered by hHGF used in our in vitro culture condi-
tion were suffi cient for the differentiation of ACA cells
along the endodermal lines indicating a great develop-
mental potential of ACA-generated cells.
The functional hearth is comprised of distinct
mesoderm-derived lineages including that of endo-
thelial cells, cardiomyocytes, and vascular smooth
muscle cells. We next performed directed differentia-
tion of ACA cells into mesodermal cell lines in cul-
tures to assess the potential of ACA cells to display
endothelial or vascular smooth muscle potential in
vitro. ACA-generated stem cells were cultured in en-
dothelial cell basal medium supplemented with fac-
tors known to support growth of these cells. Only 4
days after starting ACA-activated PBMNCs cultures
in this medium, newly generated cells can uptake
Dil-labeled acetylated low-density lipoprotein (Dil-
ac-LDL), which is a characteristic of endothelial cells
[19]. Dil-labeled ACA-derived cells were further cul-
tured and their endothelial identity was confi rmed by
morphology and immunophenotyping with antibod-
ies recognizing human vascular endothelium (VE)-
cadherin, CD31 (PECAM) and H-CAM, known for
their role in endothelial cell biology through control
of adhesion and organization of the intercellular junc-
tions (Fig. 6, c). Moreover, we found cells expressing
vascular endothelial growth factor KDR, a signaling
protein involved in the regulation of angiogenesis and
vasculogenesis known to represent vascular progeni-
tor cells defi ning the earliest stage of human cardiac
development [24]. Semi-quantitative RT-PCR analy-
sis revealed the differential expression of markers
characterizing various mesoderm-derived cell lines,
like melanoma cell adhesion molecule (MCAM) de-
tected throughout the body in endothelial cells in
vascular tissue, which plays a role in cell adhesion
Cell Technologies in Biology and Medicine, No. 2, August, 2013
561
Fig. 5. Immunophenotyping of ACA-generated pluripotent stem cells.
ACA-generated pluripotent stem cells were grown on Matrigel coated plates or in suspension and analyzed for the expression of ES cell-
specific marker.
a-c) Immunostaining of pluripotency marker in ACA-generated stem cells grown on Matrigel coated plates. DAPI staining indicates the cell
content per field. Images were taken by fluorescent microscopy.
d-g) Immunostaining of pluripotency marker in ACA-generated stem cells grown in suspension. Images were taken by confocal microscopy.
Z. A. Becker-Kojić, J. R. Ureña-Peralta, et al.
562
Fig. 6. In vitro and in vivo differentiation potential of ACA-generated pluripotent stem cells.
ACA-generated pluripotent stem cells were subjected to lineage directed differentiation and analyzed by
means of microscopic imaging, immunocytochemistry and semi-quantitative RT-PCR.
a) Histological images of ACA-generated pluripotent stem cells upon differentiation into neuronal cells.
b) Immunocytochemistry of ACA-generated pluripotent stem cells after directed differentiation into hepatic
lineages showing expression of -fetoprotein, albumin and cytokeratin 18. Nuclei were stained with DAPI.
c) Immunostaining of ACA pluripotent stem cells differentiated into mesodermal lineages expressing marker
HCAM, VE-CAD and CD31. DAPI was used to stain nuclear DNA.
d) RT-PCR analysis of genetic markers characterizing neuronal differentiation of ACA-generated pluripo-
tent stem cells.
e) RT-PCR analysis of hepatic differentiation of ACA pluripotent stem cells.
f) ACA-generated pluripotent stem cells differentiate into mesodermal lineages as judged by RT-PCR
analysis.
Cell Technologies in Biology and Medicine, No. 2, August, 2013
563
as well as the endothelial cell-specifi c molecule 1
(ESM1), expressed in endothelial cells in human lung
and kidney tissues. In consistency to our immuno-
phenotyping data for ACA-derived endothelial cells,
PCR analysis revealed that these cells express the
vascular endothelial growth factor receptor FLT1, a
receptor tyrosine kinase in VEGF signaling the most
important pathway defi ning endothelial cell mitogen-
esis and cell migration (Fig. 6, f).
In order to test our hypothesis of whether ACA-
generated stem cells at the stages with developmental
potential to evolve into all three germ layers could
differentiate in situ into environmental cells, we in-
jected unilaterally ACA undifferentiated cells in the
area of cortex and striatum of NOD/SCID mice and
analyzed thereafter. Two of three transplanted mice
were positive for human cells. PKH26 red fl uorescent
membrane labeled ACA cells were fi rstly analyzed
for the expression of human nuclei to trace the en-
grafted ACA cell in mouse brain. The frozen sections
of engrafted mouse by means of immunohistology
using antibodies, recognizes human antigens as well
as human neuronal differentiation markers (Fig. 7).
Red-labeled ACA cells, positive for human nuclei,
express vimentin, which is one of two major interme-
diate fi laments of astrocytes and glia appearing early
in the neural development. Vimentin is transiently
expressed in many cells of neuroectodermal origin
in the fetal central nervous system and proliferation
of progenitor cells in the adult rat brain correlates
with the presence of vimentin expressing astrocytes
[1]. The majority of ACA-engrafted cells expressed
vimentin (Fig. 7, b) indicating that an early stage of
neural differentiation occurred through environmen-
tal signals delivered to undifferentiated ACA cells by
surrounding mouse brain cells, thus providing a pool
of proliferating neural progenitor/stem cells with ca-
pability to differentiate into more specialized types
of brain cells. Some of the engrafted red-labeled cells
expressed marker characteristic for highly specialized
cells, like N-CAM (Fig. 7, e) usually expressed in
neurons and implicated in neurite outgrowth, synap-
tic plasticity, learning and memory. Our data dem-
onstrate the capability of ACA cells to distinctive
differentiation probably driven through signals de-
rived from microenvironment in concordance with
data shown previously (Becker-Kojić Z. et al., sub-
mitted), that intravenously injected, undifferentiated
ACA pluripotent stem cells, matured in bone marrow
of NOD-SCID mice to progenitor/HSCs in brain to
vimentin/N-CAM cells and engrafted other organs
like spleen, liver, kidney and lung.
In conclusion, these fi ndings raise one main is-
sue – does the unique progenitor cell exist during the
development and does the location control the speci-
fi cities between progenitors in different germ layers
designating the origin and fate of these cells?
DISCUSSION
The discovery that somatic cells typically exhibit little
or no capacity to differentiate and that they can be
reprogrammed to obtain iPSCs capable of forming
any cell type of the body has been proven as a break-
through in stem cell biology. Despite the signifi cance
of the new information provided many questions sur-
rounding iPSCs remain unanswered, preventing their
use in clinical practice.
The major drawback for using iPSCs is the lim-
ited understanding of molecular events underlying the
reprogramming mechanism, which occur via many
pathways and do not always lead to pluripotency. The
resulting cells and their progeny may constitute a high
tumorigenic risk with potential genetic damage caused
using genetic manipulation, even with improvements
in the virus-free and transgene-free reprogramming.
Teratoma forming is one of the main criteria for plu-
ripotency of embryonic stem cells (ESCs) as well as
iPSCs, but at the same time represents the main ob-
stacle for applying these cells in the clinic [4].
Our data show that ACA is expressed in all de-
velopmentally relevant human cells, like oocytes,
embryos, and ESCs as well. Considering ACA as a
signaling molecule, we hypothesize that the hot spots
with high expression of ACA in human embryo serve
as the platforms for organization and coordination of
intensive signaling crosstalk which drives the human
development, indicating a key role of ACA in human
embryogenesis. The activation by ACA ensures the
maintenance of pluripotency in human embryonic
cells. Above all, the activation by ACA in human
blood progenitor cells alone is suffi cient to induce
the signaling machinery, which leads to generation of
self-renewing pluripotent stem cells indicating a main
role of ACA in promoting self-renewal. In addition,
promoting of self-renewal in adult progenitor cells by
activating ACA specifi c signaling pathway(s) induces
pluripotency in these cells.
Our results show that the pluripotent stem cells
can be generated without any form of genetic manipu-
lations suggesting an intriguing and advantageous al-
ternative to the use of ES or iPS cells. ACA-generated
stem cells are pluripotent by their genetic markers and
differentiation potential, but like all other adult stem
cells, they do not form teratoma.
The data provided in this work permits us to pro-
pose the mechanism of action of ACA, which leads to
a generation of ACA pluripotent stem cells.
Upon ACA antibody crosslinking at the surface
of blood progenitor cells ACA induces signals, which
Z. A. Becker-Kojić, J. R. Ureña-Peralta, et al.
564
give rise to their pluripotency via PI3K/Akt/mTOR
signaling.
The phosphorylation of PLCγ and its activation
is the crucial event in ACA signaling pathway(s) and
indispensable for inducing pluripotency in progenitor
cells. This enzyme hydrolyses ACA protein triggering
the mechanism responsible for generation of signaling
molecules known as second messengers, like diacylg-
lycerol (DAG), phospatidylinositol phosphates which
are phosphorylated through action of class 1 PI3 ki-
nase, a key enzyme in signal transduction from various
stimuli to down-stream pathways. This reaction further
includes activation of PDK1, the transducer of PI3K
action by phosphorylation of Akt, which is central for
promoting cell growth, GSK-3, a multi-tasking serine/
threonine kinase, known to regulate critical cellular
functions such as structure, gene expression and apop-
tosis, as well as mTOR complexes, a critical regulator
of translation and protein synthesis. The second mes-
sengers, generated upon enzymatic activity of PLCγ
on ACA, are responsible for the activation of numbers
of signaling molecules like c-Kit, Notch, Wnt, Bmi-1
as well as the homeobox transcription factors HoxB4,
HoxA4, HoxA9, and Hox10 as described previously
(Becker-Kojić Z. et al., submitted) and this explains
the mechanism by which ACA promotes self-renewal
and stem cell growth.
Most importantly, opposite to current methods
available for the generation of iPS, there is no evident
risk of tumorigenic transformation of ACA pluripotent
stem cells, because many of the factors responsible for
controlling and preventing oncogenic outgrow are ac-
tivated through ACA/IP3K/Akt signaling mechanisms.
TGF-β, an inhibitor of HSCs growth and anti-apoptotic
gene BCL-2, are down-regulated in ACA-specifi c sig-
naling cascade. PTEN, tumor suppressor gene acting
as a lipid phosphatase, is a negative regulator of one
of the best conserved functions of Akt by preventing
rapid growth and division of cells [8] is up-regulated
in an ACA-dependent manner. In addition, tumor sup-
pressor antigen p53 [13] remains constant or slightly
up-regulated throughout reprogramming by ACA (data
not shown).
Overall, the study results suggest that activation
by ACA promotes pathway(s), which enable cells to
cross the lineage boundaries and differentiate to cells
of the other germ layers. Accordingly, ACA stem cells
altered its pattern of gene expression, and consequent-
ly its fate, to that typical of a cell type of another germ
layer.
The method, which leads to generation of HSCs,
SP, as well as pluripotent stem cells, suggests that the
unique principle exists relaying on activation of ACA
by inducing up-regulation of evolutionary conserved
c-Kit, Wnt, Notch signaling pathways via PI3K/Akt/
PTEN/mTOR, resulting in the regulation of self-
renewal in stem cells. Therefore, the generation of
HSCs, SP and pluripotent stem cells are different steps
along a continuum, representing a process initiated
through ACA, possibly responsible for replenishment
and maintenance of stem cell pool in human body.
Fig. 7. ACA-generated pluripotent stem cells differentiate in vivo
into neural precursor cells.
Immunohistology of frozen section of engrafted mice transplanted
with PKH 26 labeled ACA-generated pluripotent stem cells. DAPI
was used to visualize nuclear DNA.
a) A schematic overview of in vivo transplanted ACA-generated
pluripotent stem cells
b) Human nuclei antibody is used to characterize engrafted hu-
man cells.
c) Transplanted cells express human GPI-linked glycoprotein ACA.
d) ACA pluripotent stem cells differentiate into neural precursor
cells expressing vimentin.
e) Engrafted ACA pluripotent stem cells differentiate into neural
marker cell N-CAM.
Cell Technologies in Biology and Medicine, No. 2, August, 2013
565
ACA-generated cells show a pluripotent devel-
opmental potential without the imperative of reaching
the stage of pluripotency. The generation of neuro-
nal, hepatic or endodermal cells was obtained at days
8-12, the time when the cells no longer expressed the
hematopoietic marker CD34. They resembled phe-
notypically SP cells that are known to have a greater
differentiation potential than HSCs. This could have
an additional important advantage, since reducing the
extent by which the cells need to be reprogrammed,
could reduce the potential genetic damage.
Blood cells represent an easily accessible source
of cells that obviate the need for skin biopsies com-
monly applied in current methods for derivation of
iPS and require minimal maintenance in culture be-
fore reprogramming. All immature cells present in
blood at all stages of differentiation are reprogrammed
under the same manner defi ned through ACA/PI3K/
Akt/PTEN/mTOR pathway. Therefore, ACA-generated
cells need not to be germ line or even teratoma com-
petent in order to be approved of accomplishing the
process of reprogramming, as long as they can self-
renew and produce useful progeny. At all stages of de-
differentiation, ACA cells have a certain differentiation
potential, the more primitive the pheno- and genotype,
the greater the differentiation potential. Hence, whether
ACA reprogrammed cells can cross the lineage restric-
tions and differentiate into the cells of other germ layers
is entirely the matter of the level of de-differentiation
regulated by signaling machinery initiated by the acti-
vation of ACA specifi c signaling network.
Overall, our studies signify that ACA, as a mem-
brane receptor is involved in developmentally conserved
signaling pathway(s) known to shape the structure of
embryo, but infl uencing somatic stem cell compart-
ments as well, indicating that the universal pathway(s)
regulating all these processes must exist in vivo. Here,
we identifi ed a molecular mechanism, which might be
able to provide the human body with stem cells having
appropriate developmental potential needed in the case
of injury. Receptor occupancy of ACA through antibody
crosslinking implies that its activity must be regulated
in vivo, probably through small molecules, e.g. soluble
natural ligand(s). Stem cells microenvironment known
as stem cell niche might provide ACA’s ligand(s). Bind-
ing of natural ligand(s) to ACA receptor and initiation
of ACA signaling network is likely to determine the
level of de-differentiation and consequently the fate of
stem cells, therefore controlling the balance between
renewal and differentiation [16].
As blood fl ow is available in all organs, blood
progenitors could be reprogrammed and differenti-
ated upon stimulation due to different location in the
body providing that injury, repair, and replacement of
diseased cells, e.g. regeneration of adult tissue might
exploit the same ACA molecular pathway(s) probably
used to establish these tissues during development.
Soluble ACA ligand(s) might be in addition to
ACA-specific antibody a highly desirable tool(s),
which could help omit cytotoxic preparative regiments
explored usually for in vitro transfection conditions
used thus far for generation of pluripotent stem cells.
ACA cells provide a real therapeutic alternative to
the controversial use of ESCs or iPSCs or therapeu-
tic cloning and may enhance tissue regeneration via
ACA’s mechanism to a clinically useful level. Above
all, we have shed the light on the molecular events,
which occur during early development, and opened
up a new avenue for studying the developmental bi-
ology in humans. ACA undifferentiated pluripotent
stem cells will be the most valuable tool for future
studies of homeostasis, tissues regeneration and cell
replacement having profound implication for our un-
derstanding of fundamental processes occurring in the
human body.
EXPERIMENTAL PROCEDURES
Cell cultures
ACA pluripotent stem cells were generated from hu-
man PBMNCs through ACA crosslinking with its spe-
cifi c antibody and the generation of ACA cells in the
presence or absence of inhibitor (ET, LY, or ET+LY)
performed in Iscove’s modifi ed Dulbecco’s medium
supplemented with 10% FBS. Cells were taken at dif-
ferent time points for immunophenotyping by fl ow
cytometry. Pluripotent ACA stem cells were cultured
in suspension in Iscove’s medium or on mouse embry-
onic feeder cells (MEF) in ESC medium. Feeder-free
culturing of ACA pluripotent stem cells was performed
on culture dishes coated with Matrigel using condi-
tion media in order to assess the expression of hESC
antigens by means of immunofl uorescence.
Cells from primary hESC line H9 were grown
on fresh commercially available human foreskin fi -
broblast inactivated with mitomycin C in Knockout-
DMEM medium.
Immunocytochemistry
ACA-specifi c monoclonal antibody was used to ana-
lyze the expression of ACA in fi xed human oocytes
and 1, 2, 3, and 5 days post in vitro fertilized embryos
obtained from University Hospital La Fe Valencia,
Spain. Goat anti-mouse Alexa 488 conjugated antibody
(Molecular Probe) was used for secondary labeling ac-
cording to suppliers instruction. Primary antibodies for
characterization of hESC cell line H9, ACA-derived
pluripotent stem cells or differentiated ACA cells were
typically applied for 1h, washed three times with PBS
for 5 min and incubated with appropriate Alexa-Fluor
Z. A. Becker-Kojić, J. R. Ureña-Peralta, et al.
566
488 or Alexa 568 (Molecular Probe) conjugated sec-
ondary antibodies for 30 min. After washing, the cells
were mounted on slides with ProLong® Gold antifade
reagent containing DAPI (Invitrogen).
Flow cytometry analysis
The expression of activation, lineage associated anti-
gens and hESC antigens in ACA-generated stem cells
were assessed by time course fl ow cytometry using
FACS Calibur (Becton-Dickinson) or FACS Cytomic
FC 500 (Beckman-Coulter fl ow cytometer). Conju-
gated isotypes-matched irrelevant monoclonal antibo-
dies (mAbs) were used as controls. Gating was done
with matched isotype control mAbs. The nonviable
cells were excluded through staining with propidium
iodide (PI).
Western immunoblotting
Phosphorylated and total primary antibodies were pur-
chased from Cell Signaling Technology. GAPDH an-
tibody was obtained from Santa Cruz Biotechnology.
Western blot analysis was performed using standard
techniques. In brief, ACA-generated cells were lysed in
Triplex buffer (50 mM Tris HCl pH8, 120 mM NaCl,
0.1% SDS, 1% Nonidet P-40, 0.54% deoxycholate.
Equal amount of total lysate, 30 μg proteins per sample
were analyzed by 10% SDS-polyacrilamide gel electro-
phoresis, and after blotting, membranes were incubated
overnight at 4oC with primary antibodies, followed by
HRP-conjugated antibody. The targeted protein was
revealed using Pierce enhanced chemiluminescence kit
according to supplier’s instruction. Protein preparations
of non-treated PBMNCs were used as controls.
Semi-quantitative RT-PCR analysis
The expression level of pluripotent, or differentiation
genes were assessed by semi quantitative PCR analy-
sis. Total RNA from ACA-generated cells was extract-
ed using RNA Isolation IKit (Zymo Research, Orange,
CA). After DNase treatment cDNA was isolated using
Taqman Reverse Transcription Reagents (Applied Bio-
systems) according to manufacturer’s instruction. The
cDNA concentrations were normalized in all samples
based on RT-PCR of GAPDH gene.
In vitro differentiation of ACA stem cells
For neuronal differentiation, ACA- generated cells
at day 8-10 were placed in ornithine/laminine-coated
plates (Invitrogen) and cultured in GRM medium ac-
cording to the methods [6].
For hepatic differentiation ACA-generated stem
cells at day 8-10 were grown onto gelatin coated plates
(0.1% gelatin in PBS (Invitrogen) as described in [11]
or in DMEM supplemented with hepatocyte growth
factor (HGF, 10 ng/ml).
For mesodermal differentiation ACA cells were
grown on fi bronectin coated plates and cultured in
endothelial cell basal medium (EBM-2 Basal medium
Lonza CC-3156) supplemented with EGM-2 MV sin-
gle aliquots (Lonza CC-4176).
ACA-differentiated neuronal, mesodermal, and
hepatic cells at different culture time points were taken
for RT-PCR or immnunophenotyping analysis.
In vivo differentiation of ACA
pluripotent stem cells
ACA-generated pluripotent stem cells at day 20 and
day 22 respectively were labeled with PKH26 Red
Fluorescent Cell Linker Kit (Sigma) and ca 6x105
cells were transplanted in anesthetized (1.5-2% Iso-
fl urane) mice (NOD/LtSz-Scid IL2Rγnull) positioned
under a stereotaxic apparatus (Stoelting). ACA cells
were injected unilaterally in one point, corresponding
to the cortex and striatum. The 100-120 nl volume
were injected in each of the 6 different points of the
dorsal axe at the following coordinates from Bregma
(L: ±1.5; A: +0.14; D: -2.8/-2.3/-1.8/-1.3/-0.8/-0.5),
according to the compact mouse brain atlas (Paxinos
and Franklin, 2004). Control sterile PBS solution was
injected performing the same surgical procedure in
the contra lateral brain hemisphere applying the same
volume and stereotaxic coordinates. After surgical pro-
cedure, grafted mice were returned to their cages and
analyzed 3 months later.
ACKNOWLEDGMENTS
We are grateful to Dr. Annie Schott for her encourage-
ment and permanent support in the most trying time.
We thank to all our volunteers for blood donations and
Isabel Roglas for drawing the blood. We also would
like to thank the confocal microscopy unit at CIPF for
excellent assistance with microscopic imaging, Dr. C.
Dufter for his assistance with FACS analysis and Anja
Dobrilović for editing the manuscript.
ACA specifi c antibodies are patented for applica-
tions related to generation and expansion of haemato-
poietic and pluripotent stem cells.
REFERENCES
1. G. Alonso, Glia, 34, No. 4, 253-266 (2001).
2. F. Anokye-Danso, C. M. Trivedi, D. Juhr, et al., Cell Stem
Cells, 8, No. 4, 376-388 (2011).
3. Z.A. Becker-Kojić, Use of ACA glycoprotein antibodies for
obtaining/maintainig pluripotent non-embryonic stem cells.
EP 1593737.A1.(2005).
4. J. L. Cox and A. Rizzino, Exp. Biol. Med. (Maywood), 235,
No. 2, 148-158 (2010).
5. B. W. Doble and J. R. Woodgett, J. Cell Sci., 116, Pt. 7, 1175-
1186 (2003).
Cell Technologies in Biology and Medicine, No. 2, August, 2013
567
6. S. Erceg, S. Laínez, M. Ronaghi, et al., PLoS One, 3, No. 5,
e2122 (2008).
7. M. J. Evans and M. H. Kaufman, Nature, 292, 154-156 (1981).
8. L. He, A. Ingram, A. P. Rybak, and D. Tang, J. Clin. Invest.,
120, No. 6, 2094-2108 (2010).
9. R. Jaenisch and R.Young, Cell, 132, No. 4, 567-582 (2008).
10. K. Kaji, K. Norrby, A. Paca, et al., Nature, 458, 771-775
(2009).
11. S. Kakinuma , Y. Tanaka, R. Chinzei, et al., Stem Cells, 21,
No. 2, 217-227 (2003).
12. S. V. Liu, Stem Cells Dev., 17, No. 3, 391-397 (2008).
13. R. M. Marión, K. Strati H., Li, et al., Nature, 460, 1149-1153
(2009).
14. I. H. Park, R. Zhao, J. A. West, et al., Nature, 451, 141-146
(2008).
15. G. Paxinos and K.B.J. Franklin, The Mouse Brain in Stereo-
taxic Coordinates. Amsterdam (2004).
16. D. T. Scadden, Nature, 441, 1075-1079 (2006).
17. R. Sridharan and K. Plath, Cell Stem Cells, 2, No. 4, 295-297
(2008).
18. K. Takahashi, K. Tanabe, M. Ohnuki, et al., Cell, 131, No. 5,
861-872 (2007).
19. O. M. Tepper, R. D. Galiano, J. M. Capla, et al., Circulation,
106, No. 22, 2781-2786 (2002).
20. J. A. Thomson, J. Itskovitz-Eldor, S. S. Shapiro, et al., Science,
282, 1145-1147 (1998).
21. J. Wang, S. Rao, J. Chu, et al., Nature, 444, 364-368 (2006).
22. K. Woltjen, I. P. Michael, P. Mohseni et al., Nature, 458, 766-
770 (2009).
23. G. Xu, W. Zhang, P. Bertram, et al. Int. J. Oncol., 24, No. 4,
893-900 (2004).
24. L. Yang, M. H. Soonpaa, E. D. Adler, et al., Nature, 453, 524-
528 (2008).
25. J. Yu, M. A. Vodyanik, K. Smuga-Otto, et al., Science, 318,
1917-1920 (2007).
26. T. Zhao, Z. N. Zhang, Z. Rong, and Y. Xu, Nature, 474, 212-
215 (2011).
Z. A. Becker-Kojić, J. R. Ureña-Peralta, et al.
