Vol.3, No.4, 240-250 (2013) Stem Cell Discovery
http://dx.doi.org/10.4236/scd.2013.34029
Embryonic-like stem cells derived from postpartum
placenta delivered after spotaneous labor emerging
as universal prophylactic cancer vaccine
Manole Corocleanu
Private, Brasov, Romania;
Received 19 October 2013; revised 25 October 2013; accepted 30 October 2013
Copyright © 2013 Manole Corocleanu. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
In the eighth decade of the last century exten-
sive clinical delayed-type hypersensitivity (DTH)
skin tests to an intradermal injection of a phar-
maceutical allogeneic human Placenta Suspen-
sion (phPS) performed in obstetrical, gynecolo-
gical and control group patients have shown po-
sitive reaction in 239 patient s with clinical condi-
tions having been as histopatrhological substra-
tum, a hypoxia-induced adaptiv e/reactive epithe-
lial cell proliferation, e.g. syncytiotrophoblastic
cell hyperplasia, endometrial cell hyperplasia, or
different gynecological cancers. Because the im-
mune response against phPS has shown anti-
genc similarities between normal placental and
endometrial hyperplastic cells and different kinds
of cancer cel ls and becau se many can cers adopt
an embryonic stem-like gene expression pattern,
it is suggested that the profile of hypoxia-pro-
moting placental and endometrial stem cell pro-
liferation is more embryonic-like, and that the
immune respose against phPS is expected to
cross-react with tumor cells in vivo. In the pro-
cess of persistent growth and accelerated oxy-
gen consumption by hyperplastic cytotrophoblas-
tic cells and neoplasti c cell s in a hypoxi c micr o-
environment, a basic shift in energy metabolism
is accompanied by appearance of heat shock
proteins (HSPs), of fetal isoenzymes and of mem-
brane glycoproteins (reappearance of oncofetal
antigens, OFAs), w hich, as result of their overex-
pression/amplification may induce a host immu-
nological response. Thus, it is assumed that
phPS prepared from full-term human placentas
delivered after a spontaneous labor comprises
stem/progenitor cells reverted to a proliferative
embryonic stem cell-like-state upon exposure to
labor-inducing intrmittent placental hypoxia and
that by expressing HSP/OFAs could immunize to
generate immune response againjst a variety of
antigens that are shared by different kinds of
epithelial cancers. This immunological feature of
phPS qualifies is as a vaccine-related product
that may be used for a preventive cancer vac-
cine when mixed with a potent adjuvant (BCG-
Vaccine) and given normal healthy individuals.
Keywords: Placenta; Embryonic-Like Stem Cells;
Preventive Cancer Vaccine
1. INTRODUCTION
The field of tumor immunology has made great ad-
vancements in recent years. A retrospective analysis of
our previous studies [1] and our proposed pharmaceutical
allogeneic human Placenta-Lysates/BCG-vaccine strat-
egy design for cancer prevention [2], based on the as-
sumption that placenta shares identical growth mecha-
nisms, antigenic determinants, and immune-escape prop-
erties with cancer cells, combined with present knowl-
edges may provide additional insight in this vaccination
approach.
Proteins that are expressed by both malignant and
healthy fetal tissues (the placental-fetal complex) are
recognized as oncofetal antigens (OFA). These antigens
are associated with cell proliferation and differentiation
and are produced in high concentrations in pregnancy
and malignancy [3]. Their biological role in malignancy
is the suppression of the host’s immune system, while in
pregnancy they affect the maternal immune response,
generating maternal tolerance toward the embryo [4]. On
the other hand, the involvement of oncofetal antigens in
both embryonic and malignant development supports the
concept that oncofetal antigens may intervene in the con-
trol of maternal immune responses during pregnancy, in

M. Corocleanu / Stem Cell Discovery 3 (2013) 240-250 241
the manner of the host defense to carcinogenesis [5].
1.1. Human Embryonic Genes Are
Re-Expressed in Cancer Cells and Are
Immunogenic
Marilyn Monk et al. [6] have shown that processes
occurring during tumorigenesis may be similar to proc-
esses occurring in early development. Human preim-
plantation embryonic cells are similar in phenotype to
cancer cells. Both types of cell undergo deprogramming
to a proliferative stem cell state and become potentially
immortal and invasive. The exact nature of the initiating
event is unknown but, in both processes, development
and tumorigenesis, the result is genome-wide demethyla-
tion. Therefore, it might be expected that cancer cells
will express genes in common with these very early em-
bryonic cells, especially genes specifically associated
with deprogramming and return to the undifferentiated
and proliferative stem cell state, and the maintenance of
that state.
Recently several laboratories have demonstrated that
introduction of just four active genes into a mature dif-
ferentiated cell can convert it into a cell with embryonic
stem cell characteristics [7].
Coggin Jr., J. H., et al. [8,9] has identified the OFA/
iLRP protein and its role as a T-cell inducing and im-
muno-regulating factor in fetogenesis and oncogenesis.
This protein was also detected in early to mid gestation
fetal cells and emerging trophoblast, hence the term
“Oncofetal Antigen”. It is concluded that OFA/immature
LRP, the 37 kDa fetal-restricted molecule, unlike mLRP
(mature LRP, MW 67 kDa), is a true “immunogenic.” In
breast cancer patients, Rohrer et al. established several
OFA/iLRP-specific CD4+ and cytotoxic as well as regu-
latory CD8+ T cell clones. This phenomenon caused by
an excess of the T cell immunogenic, 37 - 44 kDaOFA,
enables the immune system to suppress Tc-mediated im-
munity. In other words, it is an immuno-regulatory con-
trolled measure that prevents overproduction of Tc cells
to any Tc-antigen. This immuno-regulation prevents anti-
self Tc-mediated immunity and other anti-self immunity.
The IL-10 inhibited Tc activity and so these cells can
dampen anti-tumor immunity of whatever specificity.
During tumor development in mice and humans, onco-
fetal 32 - 44 kDa Ag/immature laminin receptor (OFA/
iLRP)-specific Th1, CTL, and IL-10-secreting T-suppres-
sor cells (Ts) cells are induced. The presence of too many
Ts or too few effector T cells appears to predict a poor
prognosis [10,11].
1.2. The Human Discarded Placentas of
Healthy Newborns Comprise
Embryonic-Like Stem Cells
Researchers at the University of Pittsburgh (2005)
have revealed findings that some placental cells of after-
birth placenta have many of the same characteristics of
embryonic stem cells and thus, placenta may yield alter-
native to embryonic stem cells.
Myoung Woo Lee et al. [12] have shown that in addi-
tion to hematopoietic stem cells (HSCs), other potential
stem cells (SCs), such as mesenchymal SCs (MSCs), un-
restricted somatic SCs (USSCs), cord blood-derived em-
bryonic-like SCs (CBEs), and umbilical cord blood (UCB)-
derived multipotent progenitor cells (MPCs) have been
isolated from Umbilical Cord Blood and characterized.
The newly discovered human cells, named “cord
blood-derived embryonic-like stem cells” or CBEs, but
also, multipotent non-hematopoietic stem cells are not
quite as primitive as embryonic stem cells, which can
give rise to any tissue type of the body, but they appear
to be much more versatile than “adult stem cells”, in that
they express some but not all embryonic stem cell markers.
1.3. The Human Endometrium Comprises
Endometrial Regenerative Cells (ERCs)
Zhong Z. et al. [13] have shown that Endometrial Re-
generative Cells (ERCs) are a population of mesenchy-
mal-like cells which possesse pluripotent differentiation
capacity and are characterized by unique surface markers
and growth factor production. They have some degree of
pluripotency similar with bone marrow derived mesen-
chymal stem cells (MSC) positive for some proteins dis-
tinctive of embryonic stem cells. Also, these endometrial
derived stem cells possess various characteristics similar
to MSC including ability to immune modulate, to induce
Treg production and to induce neoangiogenesis. Immu-
nochemical assays of cultured menstrual blood reveal
that endometrial cells express embryonic-like stem cell
phenotype markers (Oct4, SSEA, Nanong).
1.4. Hypoxia, Hypoxia-Inducible Factor (HIF)
and the Placenta
Adelman D. M. et al. [14] have shown that placental
development is profoundly influenced by oxygen (O2)
tension. Human cytotrophoblasts proliferate in vitro un-
der low O2 conditions but differentiate at higher O2 levels,
mimicking the developmental transition they undergo as
they invade the placental bed to establish the mater-
nal-fetal circulation in vivo. Hypoxia-inducible factor-1
(HIF-1), consisting of HIF-1α and ARNT subunits, acti-
vates many genes involved in the cellular and organismal
response to O2 deprivation [15].
Placental development is initiated and regulated by the
hypoxic uterine environment that is already a hypoxic,
3% - 5% O2 versus normal physiologic oxygen tension
(normoxia) of approximately 8%. O2 and the added stress
of the limited O2 diffusion stimulates trophoblasts to

M. Corocleanu / Stem Cell Discovery 3 (2013) 240-250
242
proliferate, migrate, differentiate, and promote maternal
and fetal vascular contribution to the placenta [16,17]. In
the first trimester, establishment of blood flow into the
intervillous space is associated with a burst of oxidative
stress at a time when placental vascular development is
occurring [18].
HIF-1 is a heterodimeric transcript factor that consist
of two subunits. The HIF-1 beta subunit is constitutively
expressed whereas HIF-1 alfa subunit is regulated by
oxigen levels. It is stable under hypoxic conditions but is
rapidily degraded under normoxic conditions. After sta-
bilization or activation, HIF-1 translocates to the nucleus
where it induces the transcription of numerous down-
stream genes via their hypoxia response elements. One of
the target genes is vascular endothelial growth factor
(VEGF).
Recent reports [19] suggest that preeclampsia (PE) is
associated with a Th1 predominant profile and may be
considered as a failure of the tolerance system allowing
the second physiological trophoblastic invasion. This Th1
predominant immunity is closely related to inflamemation,
endothelial dysfunction and poor placentation [10,20].
A deficiency of regulatory T cells may play a role in
the pathophysiology of preeclampsia. Immunological data
[21] have shown that the increased levels of T CD4 (+)
45RO (+) and T CD8 (+) CD25 (+) cells can suggest the
activation of CD4 (+) and CD8 (+) T lymphocytes in
pre-eclampsia. It seems possible that the activation of T
lymphocytes is associated with the deficiency of T regu-
latory cells in PE. A decreased number of T (reg) cells
were present in pre-eclampsia, and these changes might
break the maternal tolerance to the fetus.
Tereza Cindrova-Davies et al. [22] have shown that
labor is a powerful inducer of placental oxidative stress,
inflammatory cytokines, angiogenic regulators, and heat
shock proteins as result of acute hypoxia/reoxygenation
process. These findings are consistent with intermittent
perfusion being the initiating cause. Uterine contractions
during labor are known to be associated with intermittent
utero-placental perfusion. The oxidative stress is a potent
inducer of placental synthesis and release of pro-in-
flammatory factors. At cellular level the transcripts chan-
ged in the same direction as observed in preeclampsia,
suggesting that the placenta responds similarly to the oxi-
dative stress induced during labor and in preeclampsia.
1.5. Heat Shock Proteins (HSP) and Immune
Function
HSPs are evolutionarily ancient and highly conserved
intracellular molecular chaperones. They are constitu-
tively expressed in the cells, but are highly induced by
different stresses such as heat, oxidative stress, oxygen
radicals, or transformation. HSPs also perform immune
functions. They have the inherent property of binding
non-covalently to the peptides generated within cells as a
result of degradation of cellular proteins, self and alien.
HSP-peptide complexes are intracellular under normal
conditions and have a protective function. On the other
hand, extracellular located or membrane-bound HSPs
mediate immunological functions [23]. It is becoming
evident that oxidative stress is at the heart of the regula-
tion mechanisms that maintain a balance between effi-
cient regeneration and proper control of stem cell func-
tion. This activity of stem cells, however, has to be care-
fully controlled, as too much stem cell activity can cause
diseases like cancer. Cellular stress may give rise to
misfolded or mutated “self” proteins, which in turn result
in the presentation of sequences being hidden from the
immune system under normal healthy conditions. Such
hidden cryptic epitopes of “self” proteins are considered
as endogenous danger signal and also “nonself” or new
peptides by the host’s immune system because of the fact
T cells were not exposed to these hidden epitopes during
positive and negative selection in the thymus. A large
variety of chaperoned antigenic peptides could be tumor
antigens. Such HSPs are expected to carry a repertoire of
tumor antigens, some of which would be subdominant or
cryptic epitopes and seen by the host’s immune system as
nonself peptides.These consequences point to a key role
of heat shock proteins in fundamental immunological
phenomena such as activation of antigen presenting cells,
indirect presentation (or cross-priming), and chaperoning
of peptides during antigen presentation [24]. Yosino I. et
al. [25] in their research conclude that HPS70-reactive
CD4+ T cells exist in tumor tissue. These tumor-infil-
trating lymphocytes (TIL) recgnize stressed cells and
seem to play a Th1-like role that may support antitumor
T cell responses at local tumor sites.
1.6. The Role of CD40/CD40L in Regulating
Immune Response, Epithelial Cell
Growth and Differentiation and in
Induction of Cell Cycle Blockage and/or
Apoptosis of Epithelial Cancer Cells
Young L. S. et al. [26] have shown that the wide ex-
pression of CD40 in normal epithelial cells and carci-
noma cells suggests that this receptor has important, ad-
ditional influences beyond that of regulating immune
responses. The major ligand for the external domain of
CD40 is CD154 (CD40L), a member of the TNF super-
family. The most abundant source of CD154 is activated
T lymphocytes. While CD40-CD40 ligand interactions
are known to regulate B cell proliferation and different-
tiation, much less is known about the role this receptor
plays on other cell types, especially those of nonhe-
mopoietic origin. CD40 is expressed and functional on
human epidermal basal cells and that, on these cells,
CD40 ligation may be a signal for limitation of cell

M. Corocleanu / Stem Cell Discovery 3 (2013) 240-250 243
growth and induction of differentiation [27]. A direct
growth-inhibitory effect can be found when ligated
CD40 is on human breast, ovarian, cervical, bladder,
non-small cell lung, and squamous epithelial carcinoma
cells. This effect is related to the induction of cell cycle
blockage and/or apoptosis. The CD40/CD154 couple
plays a critical role both in humoral and cellular immune
response. The CD40/CD40L system, a key regulator and
amplifier of immune reactivity is required for anti-
gen-presenting cell activation as it induces costimulatory
molecules and cytokine synthesis. Thus, CD40-CD40L
interactions are crucial in the delivery of T cell help for
CTL priming. A natural antagonist of CD40/CD154 in-
teraction is the soluble form of CD40 (sCD40) which has
been shown to inhibit the binding of CD154 to CD40 in
vitro.. High levels of sCD40 could compete for the liga-
tion of membrane CD40 on CD154 thus resulting in in-
hibition of antibody production. The rapid up- and down-
regulation of CD154 on the surface of T cells is an obvi-
ous and important way of control. In a first step, CD154
is quickly expressed upon T-cell receptor engagement
and in a second step the CD40 itself contributes to down-
regulating CD154 expression on T cells as sustained in-
teraction between CD40/CD154 leads to endocytosis of
the ligand. Although this mechanism is considered to be
the major way the CD40/CD154 interaction is down-re-
gulated, the production of a soluble form of CD40
(sCD40) could also be involved, demonstrating a poten-
tial antagonistic role for sCD40 in the immune response.
However, little is known about the mechanism leading to
sCD40 production. The process of shedding is important,
as it up-regulates the production of soluble receptors
that compete with the membrane receptor for ligand
binding, and also reduces the amount of surface receptor,
thus modulating the capacity of the cell to signal and
thus, inducing immunosuppression. Given the antagonis-
tic activity of sCD40 on the CD40/CD154 interaction,
this shedding mechanism might represent an important
negative feedback control of CD40 functions [28]. As
CD40-CD154 (CD40L) pathway has been shown to at-
tribute to the regulation of T-cell activation, both by in-
dependently costimulating T cells and at least in part by
up-regulating CD80/CD86 molecules on APCs, suppres-
sion may be generated from fully differentiated Th1 ef-
fector cells by stimulation with antigen in the absence of
costimulation.
In the light of the above studies, this paper provides
further a review and a retrospective analysis in summary
of our previous published [1] and unpublished investiga-
tions to answer the question whether Placenta Suspen-
sion prepared upon Filatov’s method from the allogeneic
human placenta-tissue after a live full-term delivery, ex-
presses trophoblast cross-reactive antigens present on
certain types of trophoblast cells and on transformed
cells in the sense that both types of cells express embry-
onic-like features.
1.7. Preventive Cancer Vaccine Based on
Placental Stem/Progenitor
Embryonic-Like Cells of Full-Term
Human Placentas Delivered after
Spontaneous Labor
1.7.1. Background
Based on the assumption that developing placenta
shares identical growth mechanisms, antigenic determi-
nants, and immune-escape properties with cancer cells,
immunological cross-reactivity between placental anti-
gens and cancer antigens was investigated.
1.7.2. Methods Summary
In the eighth decade of the last century extensive
clinical delayed-type hypersensitivity (DTH) skin tests to
an intradermal injection in the 1/3 upper anterior surface
of the forearm of 0.2 ml of a pharmaceutical allogeneic
human Placenta Suspension (ph PS) (Suspensio Placen-
tae pro injectionibus, Odesski zavod Hinfarmapreparatov,
Odessa, former USSR), prepared upon Filatov’s method
from cryopreserved and mechanically disrupted of full
term human placentas delvered after spontaneous labor
were performed in obstetrical (150 pts.), gynecological
(175 pts.) patients with different clinical conditions.
All tests were made under institutional approval and
with documented informed consent.
DTH reaction is an immune function assessment that
measures the presence of activated T-cells that recognise
certain substances. Similar to the mantoux skin test for
tuberculosis, a mononuclear cell response is mounted at
the site of antigen challenge if the patient has preexisting
T cell immunity.
1.7.3. Results
239 patients with different clinical conditions, such as
hypertensive disorders during pregnancy (98 pts. with
preexisting hypertension, gestational hypertension, pre-
eclampsia, superimposed preeclampsia), abnormal peri-
menopausal and menopausal uterine bleeding (141 pts.)
have shown positive cutaneous delayed-type hypersensi-
tivity (DTH) reactions to phPS.
According to the clinical and histopathlogical diagno-
sis, two large groups have resulted in which obstetrical
and gynecological patients with different clinical condi-
tions have shown positive cutaneous DTH-response to
phPS: a group of benign obstetrical and gynecological
clinical conditions having as histopathological substra-
tum adaptive syncytiotrophoblast-cell hyperplasia (98 pts.),
or reactive/adaptive endometrial cell hyperplasia (76 pts.)
and a group of different gynecological cancers (65 pts.).

M. Corocleanu / Stem Cell Discovery 3 (2013) 240-250
244
2. CONCLUDING REMARKS
2.1. Hypertensive Disorders in Pregnancy
Stimulates Proliferation of
Cytotrophoblastic Stem Cells That
Adopt an Embryonic-Like Stem Cell
Antigenic Profile
Syncytiotrophoblast hyperplasia is commonly seen in
patients with hypertension, preeclampsia and occasion-
ally diabetes. Traditionally these clinical conditions have
been associated with “placental insufficiency”. A com-
promised maternal circulation in the intervillous space
may create a state of true or relative hypoxia that stimu-
lates proliferation of cytotrophoblast stem-like cells that
differentiate into the syncytiotrophoblast on the villosi-
ties surface in order to increase the exchange area of the
placenta. Hypoxic clinical conditions, such as arterial hy-
pertension, diabetes, but also unopposed estrogen stimu-
lation of endometrial growth are also seen in patients with
perimenopausal or postmenopausal endometrial cell hyper-
plasia which have shown positive skin reaction to phPS.
Because the immune response against phPS have
shown antigenc similarities between normal placental
and endometrial hyperplastic cells and different kind of
cancer cells and because many cancers adopt an embry-
onic stem-like gene expression pattern, it is suggested
that the profile of hypoxia-promoting placental and en-
dometrial stem cell proliferation is more embryonic-like.
Thus it is assuming that the hyperplastic cells in cyto-
trophoblastic cell hyperplasia and in abundant endo-
metrial growth, as result of perfusion-limited or diffu-
sion-limited hypoxic clinical conditions, acquire charac-
teristic traits by reactivating genes normally expressed in
emerging trophoblast and in transforming cells. It is con-
sidered that the potentialities of regeneration of the cyto-
trophoblast are possible by remodeling the “cytotro-
phoblast stem-like cells” and that the presence of “adult
stem cells” in the endometrial mesenchyme highlights
their importance in the regenesis and remodeling of en-
dometrial structures.
2.2. Labor Intermittent Hypoxia Shifts the
Antigenic Profile of Induced Placental
Proliferating Stem Cells to One That Is
More Embryonic-Like
We hypothesized also that labor intermittent hypoxia-
induced placental stem/progenitor embryonic-like cells
of afterbirth placenta could immunize to generate im-
mune response against a variety of antigens that are
shared by different kind of epithelial cancers. This raises
the exciting possibility of developing a prophylactic
vaccine capable of preventing the appearance of various
types of cancers in humans.
Hypoxia has shown to play an important role in fa-
voring the stem cell state, but also in promoting stem cell
proliferation as result of the ability of adult stem cells
capable of genomic reprogramming upon exposure to a
novel hypoxic environment to adopt the expression pro-
file to one that is more embryonic-like and express some
but not all embryonic stem cell markers (multipotent
non-hematopoietic stem cells). Also, cancer transforma-
tion is intimately coupled with the appearance” of em-
bryonic stem cell-like features, in that both overexpress
oncofetal developmental antigens (OFA) and relay ex-
clusively on glucose metabolism for their energy re-
quired for rapid cell growth and division.
Persistent growth and accelerated oxygen consumption
by hyperplastic placental cells and hyperplastic endo-
metrial cells in a hypoxic microenvironment, a basic shift
in energy metabolism is accompanied by appearance of
heat shock proteins (HSPs), increase of fetal isoenzymes
and of membrane glycoproteins (oncofetal antigens,
OFA), which, as result of their overexpression/ampli-
fication may induce a host immune response. Both types
of cell undergo deprogramming to a embryonic-like stem
cell state similar in phenptype to cancer cells. Together,
these findings suggest that HIF targets may act as key
inducers of a dynamic state of stemness in pathologic
conditions and that tissue regeneration after injury ap-
pears to recapitulate the pathway of embryonic tissue de-
velopment.
The up-modulation of OFA gene expression in hyper-
plastic placental and endometrial cells and in tumors
might be related to the requirement of proliferating cells
for increased protein synthesis to face the new growth
needs of these cells closely related to cell proliferation,
that classified it as oncogene and that by overexpres-
sion/amplification becomes immunogenic.
The mechanism by which overexpression converts a
self proteine into an immunologically recognizable anti-
gen has not been completely elucidated. Most likely the
overexpressed protein either becomes more consistently
presented to the immune system or the density of pre-
sentation crosses the threshold needed for T cell stimulation.
The abundance of HSPs in the hyperplastic placental
and endometrial stem/progenitor embryonic-like cells in
response to hypoxia, or in tumor cells may make over-
expressed antigens in these proliferative cells intrinsi-
cally more cross-presentable to the host DCs for genera-
ting protective antitumor activity.
2.3. Preeclampsia Is Characterizeed by
Hypoxia-Reactivated Placental
Stem/Progenitor Cells Reverted to a
Proliferative Embryonic Stem-Like State
and a Prdominant Th1-Cell Immune
Profile
Concerning the preeclampsia, reactivation of the cyto-

M. Corocleanu / Stem Cell Discovery 3 (2013) 240-250 245
trophoblast as result of placental hypoxia and unusual
immune response to self-antigens of the emerging tro-
phoblast, suggests that proinflammatory cytokines and
vascular oxidative stress play a role in causing hyperten-
sion by activating multiple neurohumoral and endothelial
factors and that ligation of CD40 on proliferating extra-
villous trophoblast stem/progenitor embryonic-like cells,
by CD40 ligand (CD154) on CD4+ T cells, may lead to
limitation of cell growth and induction of incomplete
differentiation and inadequate invasion of the trophoblast
and thus, to ineffective vascular remodeling of the uter-
ine spiral arteries that results in insufficient placental
perfusion as well as widespread dysfunction of the ma-
ternal vascular endothelium [20].
In conclusion, it is assuming that CD4+ T cells fre-
quently recognize nonmutated “self” antigens that are
overexpressed by both hyperplastc cytotrophoblastic and
endometrial stem cells, but also growing tumor cells.
Since patients with positive skin DTH-reaction to phPS
can harbor CD4+ T cells specific for non-mutated, dif-
ferentiation antigens overexpressed by hyperplastic pla-
cental embryonic-like stem cells and neoplastic cells,
vaccination might reasonably be expected to amplify the
frequency and strength of these pre-exsiting responses or
perhaps induce some de novo reactions.
2.4. Placental Embryonc-Like Stem Cells of
Human Full-Term Placentas Delivered
after Spontaneous Labor Shares
Antigens in Common with Different
Kind of Epithelial Cancer Cells and
Thus PhPS Emerges as a Preventave
Cancer Vaccine-Related Product
Labor-induced intermittent hypoxia promotes prolif-
eration of placental stem/progenitor cells reverted to an
embryonic-like stem cell state by expressing some but
not all embryonic stem cell markers and thus, full-term
human placenta delivered after spontaneous labor (after-
birth placenta) based on proliferating cord blood-derived
embryonic-like stem cells, hypoxia-induced multipotent
non-hematopoietic stem cells mixed with other prolifer-
ating placental matrix-stem cell populations could im-
munize to generate immune response againjst a variety
of antigens that are shared by different kind of epithelial
cancers. The abundance of HSPs in the undifferentiated
state of proliferating placental embryonic-like stem cells
may make overexpressed OFA antigens in these cells
intrinsically more cross-presentable to the host DCs for
generating protective antitumor activity both in mother
and in newborn. Such a model, although attractive, re-
mains speculative. However, together these findings
suggest that the phPS could be considered as a placental
embryonic-like stem cell vaccine and that the cutaneous
DTH-reaction to phPS is a Th1-cell response to antigens
expressed by these cells and which are also cross-reac-
tive with antigens expressed by different epithelial can-
cer cells. This feature of phPS qualifies it as a multiepi-
tope vaccine-related product that may be used as univer-
sal preventive cancer vaccine. Through imparting ex-
ogenous and activating endogenous anti-tumor mecha-
nisms within normal healthy individuals by utilizing
universal, non-mutated oncofetal antigen vaccines, e.g.
phPS vaccine based on embryonic-like stem cells, the
immune system is able to destroy nascent cancerous cells
before accumulating mutational changes are occuring.
The use of overexpressed proteins, as tumor-associated
antigens yelds rational targets for specific immunopre-
vention.
In cancer prophylaxis, we need to destroy just a single
cell—the one transformed cell that may give rise to ma-
lignancy. Among tumor associated antigens are antigens
upregulated in malignant transformation e.g. oncofetal
antigens-carcinoembryonic antigen (CEA), alphafeto-
protein (AFP), growth factor receptors-Her2/neu, telom-
erase and p53. A prophylactic vaccination that involves a
number of shared antigens may represent a strength of
this vaccination approach in as much as immune recog-
nition of multiple antigens would make it less likely for
nascent tumor cells to escape immune detection and
destruction.
3. DISCUSSIONS
3.1. A Novel Hypoxic Environment Shifts the
Antigenic Profile of Adult Stem Cells to
One That Is More Embryonic-Like
A corollary of the stem cell theory of the origin of
cancer is that cancers contain the same functional cell
populations as normal tissues: stem cells, transit-ampli-
fying cells and mature cells. Cancer tissue differs from
normal tissue in that the transit-amplifying cells that do
not differentiate to mature cells (maturation arrest) accu-
mulate in cancer, whereas in normal tissue differentiate
so that they no longer divide (terminal differentiation)
[29]. Unrestained growth and accelerated oxigen con-
sumpton by proliferating stem/progenitor (transit-ampli-
fying) cells of normal tissue, or at distance from blood
vessels indicating a hypoxic microenvironment, displays
gene expression signatures characteristic of human em-
bryonic stem-like cells, or by prmoting genomic instabi-
lity drives transformation of stem/progenitor cells also
into a stable para-embryonal condition and thus, both
may be recognized as dangerous by the immune system.
The premise that cancer cells share the expression of
oncofetal antigens with stem/progenitor embryonic-like
cells and that the immune response against these antigens
is cross-protective against cancer and that the disease
only manifests if immune response are impeded in pro-
tecting against transformation, the use of overexpressed

M. Corocleanu / Stem Cell Discovery 3 (2013) 240-250
246
proteins of these stem/progenitor embryonic-like cells, as
tumor-associated antigens yelds rational targets for speci-
fic cancer immunoprevention.
3.2. The Soluble Forms of Membrane
Molecule CD40 (sCD40) May Have a
Role in Modulating Antitumor
Responses
Systemic immunity to some gynecological tumors and
to syncytiotrophoblast hyperplasia as measured by skin
delayed-type hypersensitivity response to phPS strongly
support the notion that the host immune system, harbor-
ing specific CD40+ T cells, recognizes the presence of
the reexpressed and overexpressed oncofetal antigens
(OFAs) on transformed stem/progenitor cells, or on pla-
cental stem/progenitor cells reverted to a proliferative
embryonic-like stem cell status as result of genomic re-
programming during labor exposure to a novel hypoxic
environment, and that can remain alive for days after
delivery.
Unfortunately, this proof that the immune system can
recognize spontaneous tumors, is for no lasting benefit to
the patient. It appears that it is the immune system itself
that is hindering its own activity of defense through in-
terference with inhibitory immunological checkpoints
controlling T cell activation.
The ways tumors become unrecognizable to the im-
mune system are various and numerous.
Darrasse-Jèze G., Bergot A-S., et al. [30] emphsize
that the relative activation speed of self-specific memory
Tregs (regulatorys) versus that of tumor-specific naive
Teffs (effector) at the time of tumor emergence dictates
tumor outcome. Thus, CD4+-cell responses can also
elicit not only stimulatory but also suppressive immunity.
It is becoming clear that T regs play a pivotal role in the
tumor progression and the suppression of tumor immu-
nity. Furthermore, effector/memory regulatory T cells
increased as the primary tumor progressed. Data suggest
that effector/memory Treg cells are responsible for the
loss of concomitant tumor immunity associated with tu-
mor progression [31].
Recently it was shown that T cell help for CTLs is
critically dependent on interaction between CD40L ex-
pressed by Th cells and CD40 expressed by APCs.
CD40-CD154 interactions are of central importance
and pivotal in the induction of cellular immune responses
to many antigens [26]. Several lines of evidence indicate
that CD40 signaling is part of an important pathway in T
cell-dependent antigen presentig cell (APC or DC) acti-
vation. CD40 ligation on antigen-presenting cells (APCs)
by CD40L (CD154) on CD4+ T cells was found to be
necessary to induce CD8+ T cell priming by APC. This
pathway play a critical role in the induction type-1 cyto-
kine responses of protective immunity. Dendritic cell
(DC) exist in two stages: Immature and mature. Mature
DC cells prime T cells, whereas immature DC can induce
tolerance to the presented antigens. The immature DCs
are non immunologically quiescent; they have been
shown to induce T cell tolerance in vivo through the in-
duction of T cell anergy, direct depletion of T cells, or by
generation of regulatory or suppressor cells that block
the function of other T effector T cells. For full matura-
tion and aquisition of T cell priming capacity DCs need
to be “licensed”, which can occur by receiving pro-in-
flammatory signals in the form of CD4 T cell “help”
through CD40-CD40L interaction. Adequate activation
of T cells requires multiple signals from the DC to the T
cell. MHC-peptide recognised by the T cell recptor (TCR)
on the T cell is crucial for initial activation, but will lead
to anergy or non-responsiveness without appropriate ad-
ditional costimulation provided by interaction between
CD28 on the T cell and B7.1/B7.2 on the DC. Besides
help in CD8+ T cell priming and maintenance, CD4 T
cells have been shown to recruit and activate various cell
populations into the tumor environment, provide bystan-
der mediated killing and affect angiogenesis.
Natural killer (NK) and Natural Killer T (NKT) cells
are innate immune cells critical for the first line of de-
fense against tumorigenesis. Although NKT cells possess
NK-like cytolytic activity, their activation results in rapid
production of IFN-γ and expression of CD40L, thus pro-
viding help for activation of CD40-expressing APCs and
generation of cellular and humoral immune responses.
Therefore, it has been shown that a dominant pathway
of CD4+ help is via antigen-presenting cell (APC) acti-
vation through engagement of CD40 by CD40 ligand
(CD154) on NKT cells and CD4+ T cells. CD40L is
mainly expressed transiently on activated CD4+ helper T
cells subsequent to recognition of MHC-peptide com-
plexes. It is required for antigen-presenting cell active-
tion as it induces costimulatory molecules and cytokine
synthesis. It has been suggested that in absence of a
strong “danger” signal at this time contributes to the
ability of newly forming tumors to avoid recognition by
the host immune response. Finally, priming of CD8+
cytotoxic T lymphocytes generally requires help pro-
vided by CD4+, and the CD40-CD40L interaction was
shown to be essential in the CTL priming via activated
DCs.
Also, CD40 is expressed and functional on human
epithelial cells, and on these cells, CD40 ligation may be
a signal for limitation of cell growth and induction of
differentiation [26,27]. Ligation of CD40 on cancer cells
was also found to produce a direct growth-inhibitory
effect through cell cycle blockage and/or apoptosis with
no overt side effects on normal cells. But, the natural
antagonist of CD40/CD154 interaction is the soluble
form of CD40 (sCD40) which has been shown to inhibit

M. Corocleanu / Stem Cell Discovery 3 (2013) 240-250 247
the binding of CD154 to CD40 in vitro. High levels of
sCD40, which are often over-expressed in tumors, could
compete for the ligation of membrane CD40 on CD154,
thus resulting in inhibition of antibody production, but
also in impeding T cell activation and in limitation of
growth-inhibitory effect of cancer cells by CD 154 on
CD40 T cells. The rapid up- and down-regulation of
CD154 on the surface of T cells is an obvious and im-
portant way of control. In a first step, CD154 is quickly
expressed upon T-cell receptor engagement and in a sec-
ond step the CD40 itself contributes to down-regulating
CD154 expression on T cells as sustained interaction
between CD40/CD154 leads to endocytosis of the ligand
[7]. Given the antagonistic activity of sCD40 on the
CD40/CD154 interaction, this shedding mechanism mi-
ght represent an important negative feedback control of
CD40 functions, and thus impairing of CD4 T cells in
inducing memory-effector CD8 T cells (Tc). As all DC
types are sensitive to T-cell feedback signals delivered by
activated T cells through CD40 ligand (CD40L) and at
least in part by up-regulating CD80/CD86 molecules on
APCs, suppression may be generated from fully differ-
rentiated Th1 effector cells by stimulation with antigen in
the absence of costimulation.
Barry D. Hock. et al. [32] have shown that CD40
plays a critical role in immunoregulation, suggesting that
sCD40 may have a role in modulating antitumor res-
ponses and also may be a useful prognostic marker. The
release of soluble forms of membrane molecules pro-
vides an important mechanism by which cells can either
enhance or inhibit the signals delivered by their res-
pective membrane-bound counterparts, suggesting that in
vivo release of functional sCD40 would be immunomo-
dulatory. The release of sCD40 by the immune system
and/or by malignant cells provides a potentially powerful
mechanism for regulating antitumor responses by modu-
lating the interaction of mCD40 with its ligands.
On the other hand effective activation of T cells re-
quires engagement of two separate T-cell receptors. The
antigen-specific T-cell receptor (TCR) binds foreign pep-
tide antigen-MHC complexes, and the CD28 receptor
binds to the B7 (CD80/CD86) costimulatory molecules
expressed on the surface of antigen-presenting cells
(APC). The simultaneous triggering of these T-cell sur-
face receptors with their specific ligands results in an
activation of this cell. In contrast, CTLA-4 (CD152) is a
distinct T-cell receptor that, upon binding to B7 mole-
cules, sends an inhibitory signal to T cell activation.
CD86 could be more important for initiating T-cell re-
sponses, while CD80 could be more significant for main-
taining these immune responses [33].
This interpretation assumes that CD28 functions not as
an “on-off” switch but rather as a kind of “rheostat”
which, depending on the strength and/or duration of its
engagement by B7 molecules, can display a degree of
plasticity in the intracellular signals it generates [33].
Most CD4+ T cells belong to either the Th1 or Th2
subsets. However ~10% of them do not. These so-called
T-regulatory (Treg) cells. The CTLA-4 molecules on
Treg cells bind very tightly to the B7 molecules on anti-
gen-presenting dendritic cells and B cells. Once bound,
they kill the target (by secreting perforins). Treg cells can
also kill cytotoxic T lymphocytes (CTL) and natural kil-
ler (NK) cells.
Consistent with a B7 “competition model” because
CTLA-4 has been estimated to have 50 - 100-fold higher
affinity to B7 than CD28, it is conceivable that the T reg
cells, are competitively engaging available B7 molecules
on the APCs, thereby preventing CD28 signaling, and
subsequent T cell activation in effector T cells. [34] The
co-existence of tumor specific immunity with a progre-
ssing tumor is observed in a variety of experimental
systems and remains one of the major paradoxes of
tumor immunology. It appears that it is the immune sys-
tem itself that is hindering its own activity of defense.
This behavior marks a resemblance to that of the embryo
and of cancer [35].
The above observations lead to the conclusion that re-
expression and overexpression of the trophoblast cross-
reactive antigens present on placental hyperplasic cells,
but also on neoplastic cells and continuously shedding of
CD40 (sCD40) represent an adaptive response of these
cells to natural selection pressures as a biological re-
sponse to resist immunological recognition and rejec-
tion by the host. Inhibition of the CD/E (expressing) 40
pathway by increased levels of soluble CD40 receptor
(sCD40) which reduces availability, may impede the
immune system from mounting appropriate humoral and
cell-mediated responses against precancerous cells Given
the antagonistic activity of sCD40 on the CD40/CD154
interaction, this shedding mechanism might represent an
important negative feedback control of CD40 functions,
suggesting that sCD40 may have a role in modulating a
pre-existing or de novo anti-tumor responses through
interference with inhibitory immunological check points
controlling T cell activation.
3.3. Placental Stem/Progenitor
Embryonic-Like Cells Based Cellular
Vaccine an Efficient Immuno Preventive
of Human Malignancies
To overcome these escape mechanisms and to acquire
the goal of an optimal adaptive immune response par-
ticularly to reexpression of embryonic proteins that begin
to be overexpressed in tumor cells early in their trans-
formation and which can be considered as non-self by
the immune system, it proposed a vaccination strategy
design by intradermal injection of a pharmaceutical hu-

M. Corocleanu / Stem Cell Discovery 3 (2013) 240-250
248
man allogeneic Placenta Suspension (phPS), prepared
upon Filatov’s method and admixed with BCG Vaccine
(“danger” signal) in normal healthy individuals to induce
activated/memory T effector cells (amTeffs). This cross-
priming of the tumor cells overexpressed antigen-speci-
fic response by potent APC is a major mechanism of the
developing integrated endogenous immune response,
thereby shifting the balance from tolerance to activation
induction, and/or rejuvenating functionally inferior re-
sponses of the exausted T cells.
Activated Th1 cells have longevity compared with
other activated immune cells (APCs and CTL) and are
conventionally viewed as responsible for immune mem-
ory, capable of revitalizing the immune response if a spe-
cific antigen is reencountered, including promtly func-
tion of CD40/CD40L interaction, prior to the release of
sCD40 by the immune system and/or by transformed
cells. The presence of activated/memory T effector cells
(amTeffs) at the time of tumor emergence shifts the Treg/
Teff balance toward efficient antitumor immune response.
Alternatively, or in addition, the CD4 T cells may be re-
quired for activation of macrophages and dendritic cells,
an event crucial for killing of transformed cells. This
antisupressive approach reverses the host’s immune fac-
tors that confer a survival advantage to tumor emergence
and require an intact immune system to function.
The biological adjuvant Bacillus Calmette Guerin-
Vaccine (BCG-Vaccine) takes advantage of the fact that
it is an immunogenic compound, naturally recognized as
foreign and known to induce migration of APCs to the
site of delivery. APCs responding to the adjuvant stimu-
lation are thus able to coincidentally capture and process
placental antigens present in the inflammatory milieu.
Becker Th. et al. [36] have suggested that CD40 is a
cochaperone-like receptor mediating the uptake of ex-
ogenous Hsp70-peptide complexes by macrophages and
dendritic cells. For the generation of potent T cell im-
mune responses against self-tumor Ags, Hsp70 Ag from
Mycobacterium tuberculosis was shown to induce Th1-
polarized cytokine responses and activate DCs via bind-
ing with CD40 (perhaps via promoting cross-talk be-
tween various inflammatory cells recruited at the vaccine
site). Although the Ag targeting alone, without activation
of dendritic cells (DCs), is proposed to induce tolerance,
the vaccines expressing self-tumor Ags is rendered im-
munogenic if targeted to the APCs using mycobacterial
Hsp70 Ag [36] and thus, eliciting long-term protective
CD8+ T cell-mediated memory responses effective in
killing the emerging tumor cells.
At the same time the immune response is mediated
through the release of different cytokines which can in-
fluence the synthesis and actions of one another in the
setting of an immunoregulating cytokine network, shap-
ing an environment in which the presence of active/
memory T effector cells (am Teffs) at the very time of
tumor emergence, are able to bypass the tumor immu-
nity mediated by self-specific memory regulatory T cells.
These two aspects of a preventive vaccination with mul-
tiepitope phPS/BCG-Vaccine might be useful for gener-
ating an immune response against a broad-spectrum of
cancers, thus serving as a universal cancer vaccine.
The possibility of inducing long-term protection
against tumors by vaccination at the earliest signs of its
development has the potential to cause a dramatic para-
digm shift in the prevention of tumors.
The rationale for prevention is strong because, in that
setting one deals with an immune system that is neither
impaired by tumor- and treatment-induced suppression
nor tolerant to tumor-associated antigens that have been
encountered in the absence of correct presentation and
costimulatory/danger signals. On the other hand, a future
success of cancer prevention will depend on how effec-
tively a preventive vaccination strategy simultaneously
acts on emerging pretumor cells as well as on its micro-
environment [37].
Conceptually, to avoid the regulatory T cells’ immune
suppression that develops in tumor-bearing patients, vac-
cination with a pharmaceutical allogenic human Pla-
centa Suspension prepared upon Filatov’s method, after a
“danger signal” is added e.g. BCG-Vaccine, would be
capable of eliciting immunological responses in normal
healthy individuals, not only to marker antigens shared
between the Placenta-Suspension preparation and neo-
plasia, but also by introducing a new environment of T-
cell-induced cytokines, that provide a more “complete”
immune response, to prevent tumor development far into
future, by inhibition, or by elimination of transformed
cells at their earliest manifestation. Thus, active im-
munoprevention uses the host’s immune cells and re-
quires an intact immune system to function.
Using of isolated labor intermittent hypoxia-induced
placental stem/progenitor embryonic-like cells of after-
birth placenta per se, or of phPS prepared upon Fila-
tov’s method, as a vaccine-related product for a preven-
tive cancer vaccine, warrants further investigation.
The above hypothesis is rising as a challenge to the
scientific community in the hope that it will cause an
examination of this matter.
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