Ch 21 _regeneration_of_ischemic_cardiovascular_damage_using_whartons_jelly_as_an_unlimited_source_of_therapeutic_stem_ce

1. Chapter 21
Regeneration of Ischemic Cardiovascular
Damage Using Wharton’s Jelly as an
Unlimited Source of Therapeutic Stem
Cells
Marcin Majka, Maciej Sułkowski and Bogna Badyra
Jagiellonian University Medical College, Krakow, Poland
Chapter Outline
Introduction 281
WJ-MSC Features 283
WJ-MSCs in Preclinical Trials 284
WJ-MSCs in Clinical Trials 285
Conclusions 286
Acknowledgment 287
References 287
INTRODUCTION
Cardiovascular diseases (CVDs) are the number one cause of death in the world [1]. Over 17 million people die from
CVDs annually, which comprises 31% of all deaths worldwide. Vast majority (80%) of CVDs deaths are due to heart
attacks and strokes (WHO data). Cardiac risk concerns elderly and middle-aged people with good access to medical health
care, which places CVDs as enormous health and economic problems to our societies.
Despite all the progress in modern treatment of CVDs d namely surgical therapies, including coronary artery bypass,
balloon angioplasty, and cardiac prevention d CVDs remain a chronic and progressive burden, often leaving patients with
heart transplantation necessity [1].
Experimental therapies of the future, e.g., engineered heart replacement [2] and cardiac regenerative approaches,
become most promising, with stem cells being in the spotlight of regenerative therapy.
During fetal development, totipotent morula differentiates into blastocyst with inner cell mass (ICM) surrounded by a
layer of trophoblastic cells that will give rise to the cytotrophoblast and syncytiotrophoblast of the placenta. In further
development, ICM forms epiblast and hypoblast. Latter one develops into yolk sac and allantois, whereas epiblast’s
pluripotent stem cells form embryo’s three germ layers and extraembryonic tissues, such as umbilical cord (UC)
(Figs. 21.1A and 21.1B).
UC, which originates at day 13 of the embryonic development, is formed by fifth week of fetal development and is an
essential link between fetus and mother [3]. It is composed of distinct compartments (Fig. 21.1B), starting from um-
bilical amnion-derived epithelium layer and subamnion in outer layer, two arteries and an umbilical vein in its inner part
surrounded by Wharton’s jelly matrix. Additionally, layer of adventitia or endothelium covers each vessel. Many cell
types populate UC. Depending on isolation method, UC mesenchymal stem cells (UC-MSCs), subamnion MSCs, cord
lining MSCs, UC perivascular stem cells (UCPVSCs), human umbilical vein epithelial cells (hUVECs), and Wharton’s
Jelly MSCs (WJ-MSCs) can be distinguished [4e8]. Moreover, UC blood cells (UCBCs) can be isolated freshly after
Perinatal Stem Cells. https://doi.org/10.1016/B978-0-12-812015-6.00021-2
Copyright © 2018 Elsevier Inc. All rights reserved.
281

2. childbirth through umbilical vein. All of those cells have the advantage of being defined as “young” stem cells, and
despite their primitive character, they can be isolated without ethical concerns, as developed from extraembryonic
tissues. Moreover, those cells are acquired from medical waste after delivery and are not connected with invasive and
painful procedures. However, differences between cells obtained from separate compartments were noted, which may
suggest their distinct origin [9,10]. Among those cells, WJ-MSCs have been marked as most feasible for cellular
therapies. Ease of isolation, high yield, purity and proliferation rate make them attractive source for transplantation
procedures.
WJ is an extracellular matrix (ECM) proteinerich compartment of UC, which is composed of collagens, glycos-
aminoglycans, and hyaluronic acid [11]. Main role of such structure is to protect UC vessels from pressure, bending,
and torsions during pregnancy. Enriched ECM is also great “storage” of growth factors such as acidic and
basic fibroblast growth factors (aFGF, bFGF), epidermal growth factors (EGF), insulin-like growth factor 1 (IGF-1),
platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-b) that can support dispersed stromal
cells [12].
There are two possibilities how MSCs are populating WJ. First one claims that during fetal MSCs migrations during
development, a portion of cells can be trapped in WJ [13]. Second theory postulates that MSCs can originate
from primitive mesoderm that is present in the UC from the beginning, where their key role is to produce matrix
proteins [14].
(A) (B)
(C)
FIGURE 21.1 Photography of human umbilical cord (UC) (A). Schematic cross section of human UC (B). Microphotography of in vitro cultured WJ-
MSCs. Magnitude 100Â.White bar represents 100 mm (C).
282 SECTION j III Umbilical CordeDerived Cells

3. WJ-MSCs can be isolated from WJ enzymatically by collagenase, trypsin, hyaluronidase, or mixture of them. They can
also migrate directly from UC explants. Importantly, depending on chosen isolation method, cells can slightly differ in
features such as expression of pluripotency markers and cell proliferation rates [15].
WJ-MSC FEATURES
Important drawbacks of using adult stem cells include their naturally low number, difficulties with harvesting, and low
expansion potential. Stem cells harvested from perinatal tissues (such as cord blood, amniotic fluid, and cord) possess
several attractive characteristics that resemble those of embryonic stem cells in absence of the embryonic stem cells
disadvantages. Today, the most studied type of perinatal stem cells are hematopoietic stem cells isolated from cord blood
during delivery. Cells from the cord, however, are still underevaluated and thus underappreciated.
WJ-MSCs share common characteristics with MSCs isolated from other sources, such as adult bone marrow (BM-
MSCs) and adipose tissues (AT-MSCs). They are adherent, fibroblast-like cells, and as no separate specific marker for
MSCs has been yet described, International Society for Cellular Therapy established a gold standard panel of surface
markers to describe their phenotype [16]. Cultured MSCs are defined as cells positive for CD73, CD90, CD105 (all >95%)
and negative for CD34, CD45, CD11b/integrin alpha M or CD14, CD79 alpha or CD19, HLA class II (all <2%).
However, the latter markers can change when MSCs are exposed to inflammatory cytokines [17]. Yet, because of absence
of costimulatory CD80, CD86, CD134, CD252, MSCs are unable to induce T cell activation [17]. Moreover, when
cultured in vitro, they can also differentiate to adipocytes, chondrocytes, and osteocytes. WJ-MSCs are also capable to
transdifferentiate in vitro to cells outside mesenchymal lineages, such as cardiomyocytes, endothelial cells, and cells from
neuronal lineage [8,18], which suggests their broader-than-multipotent potential.
Yet, thanks to their origin, WJ-MSCs are presented as “young” or primitive cells in comparison with MSCs isolated
from adult sources and have superior biological properties. WJ-MSCs demonstrate features intermediate between ESCs
and adult MSCs because of expression of POUF1, SOX2, NANOG, LIN28, SSEA1, SSEA3, SSEA4, KLF4, c-MYC,
CRIPTO, and REX1 [19,20]. However, distinct lower expression of pluripotent genes in comparison with hESCs may be a
reason why WJ-MSCs do not form teratomas when transplanted to immune-deficient mice [21]. Unlike BM-MSCs, WJ-
MSCs are not able to transform to tumor-associated fibroblasts (TAFs) phenotype, when exposed in vitro to breast or
ovarian cancereconditioned media [22]. Furthermore, WJ-MSCs display anticancer properties, as shown as elevated
expression of IL-12 or cancer growth inhibition in xenograft model [20,23].
Accordingly, thanks to origin of WJ-MSCs, these cells have profound immune tolerance and are even called hypo-
immunogenic cells. Physiologically, it may be related to their presence in the UC and prevention of fetus rejection. It is
based on secretion of a variety of factors, e.g., HLA-G and CD274 [17]. Notably, high expression levels of HLA-G6 are a
unique feature of WJ-MSCs [24]. It was also shown that WJ-MSCs have the lowest expression of HLA-DR even in
immune-primed conditions among MSCs [25]. Also, WJ-MSCs can secrete higher levels of hepatocyte growth factor
(HGF), prostaglandin E2 (PGE2), and decreased amount of insulin-like growth factorebinding protein-3 (IGFB3) because
of inflammatory cytokines stimulation [17]. Moreover, WJ-MSCs can diminish immune responses by secreting key
regulatory factors such as IL-10, TGF-b, IL-6, vascular endothelial growth factor (VEGF), indoleamine 2, 3-dioxygenase
(IDO), and B7eH1 [24,26e28]. WJ-MSCs were also shown to uniquely secrete IL-12, IL-15, and PDGF-AA [26].
Functionally, WJ-MSCs can greatly suppress allogeneically stimulated T lymphocytes and inhibit maturation and acti-
vation of dendritic cells (DCs) [26,28].
Substantial immune tolerance plays a crucial role in choosing WJ-MSCs as a perfect source for allogeneic trans-
plantations, especially in acute cardiovascular disorders when transplantation is needed immediately after occurrence of
injury because of lack of time for propagation of autologous MSCs. Allogeneic transplantation also solves problems of
limited amount of autologous MSCs or their altered phenotype because of CVDs’ co-morbidities [29]. However, it is
noteworthy that detailed analysis of mother’s health is needed, as conditions such as diabetes can influence WJ-MSCs’
performance [30].
WJ-MSCs in comparison with other MSCs express key early cardiac transcription factors, such as Flk-1, Isl-1, Nkx2.5,
GATA-4, GATA-5, and GATA-6, which can be significant for therapies of CVDs [19,31]. WJ-MSCs can secrete a
plethora of factors, which were reviewed elsewhere, and their secretome was analyzed in comparison with other sources
[32e34]. Briefly, WJ-MSCs secrete many proangiogenic factors such as VEGF, HGF, angiopoietin and TGF-b1, that are
critical for cardiac remodeling and progression after heart failure and can also influence resident cardiac stem cells [32,35].
However, BM-MSCs and AT-MSCs were shown to have more profound proangiogenic character [32]. WJ-MSCs have
high expression of CD54 and CD146dadhesion molecules that are related to their migratory character.
Wharton’s Jelly MSC and Cardiovascular Damage Chapter | 21 283

4. WJ-MSCs can also differentiate into cardiomyocytes by use of 5-azacytidine, oxytocin or through “embryoid body”
formation [36]. However, oxytocin seems to be a more potent inducer of differentiation, which is consistent with the fact
that this peptide is highly expressed in the developing heart [37]. Differentiation of WJ-MSCs can be assessed by
expression of cardiac troponins, connexin 43, as well as changes in morphology. However, the differentiation of WJ-MSCs
to cardiomyocytes in vivo is still debatable, as paracrine effect of these cells has a profound regenerative potential [38].
WJ-MSCS IN PRECLINICAL TRIALS
There are numerous reports proving highly beneficial effects of BM-MSC transplantation in animal models of CVDs
[39e45]. However, there are as many limitations of their use, which involve, but are not restricted to, invasiveness of
acquisition, age of donor, limited cell number. WJ-MSCs possess unique features that make them ideal candidates for
regenerative therapies of the heart, even among MSCs of different origin.
WJ-MSCs, like other MSCs, have the potential to differentiate into key cells lost in heart infarction d cardiomyocytes.
WJ-MSCs treated in vitro with 5-azacytidine- or cardiomyocyte-conditioned medium expressed cardiomyocyte markers,
namely, N-cadherin and cardiac troponin I [8], suggesting cardiac differentiation. This ability was also observed for
perivascular MSCs in the UC [46] but interestingly not for UC blood cells [47,48].
WJ-MSCs were shown to be capable of cardiac function improvement in the rat model of myocardial infarction [49].
WJ-MSCs injected into infarcted regions survived for as long as 4 weeks and were found around arterioles and scattered in
capillary networks. Transplanted cells expressed cardiac troponin-T, von Willebrand factor, and smooth muscle actin,
indicating that multilineage differentiation of WJ-MSCs d into cardiomyocytes, endothelial cells, and smooth muscle cells
d takes place during regeneration of ischemic myocardium. More importantly, parameters of the heart improved upon WJ-
MSC transplantation. Left ventricular ejection fraction (LVEF) and left ventricular posterior wall thickness improved, with
a decrease in apoptotic cells [49]. Moreover, capillary and arteriole density increased compared with the control group,
confirming the angiogenic potential of WJ-MSCs. It was previously shown that WJ-MSCs can differentiate into endothelial
cells expressing endothelial-specific proteins, such as platelet endothelial cell adhesion molecule, PECAM, and CD34 both
in vitro (when stimulated with VEGF and bFGF) and in vivo (endothelial cells sprouting from local injection in a hindlimb
ischemia mouse model) [50]. What is noteworthy is that similarly beneficial effects delivered by UC perivascular cells [46]
increased cardiac function in murine model of acute myocardial infarction 14 days after transplantation.
Several weeks may seem to be not long enough for drawing unambiguous conclusions concerning efficacy of WJ-
MSCs transplantation; nevertheless, WJ-MSCs were also proved to have long-term beneficial effects on infarcted heart
in rat model [51]. Intravenous injection of WJ-MSCs proved not only homing capacity of these cells toward injured
sites (cells were found in periinfarction regions) but also significant capacity for prolonged improvement in ejection
fraction. These improvements were observed in treated animals for at least 31 weeks. Homing and integration of WJ-MSCs
with cardiac tissue were also described previously [52]. WJ-MSCs were markedly chemoattracted toward the embryonic
murine ventricular slices and were able to integrate robustly into the depth of both living and ischemic cardiac tissues
in vitro.
Another study showed that WJ-MSCs injected into murine heart directly after myocardial infarction induced by left
anterior descending coronary artery ligation preserved heart function and attenuated cardiac remodeling process, evaluated
2 weeks after the infarction. They also stimulated angiogenesis that increased capillary density; however, there were no
differences in infarct size upon cells transplantation. Apoptosis in the infarcted tissue was also prevented. Moreover,
WJ-MSC-conditioned medium was able to enhance cellular vasculogenesis and activate cardiomyogenic gene expression
program in vitro, proving strong paracrine activity of WJ-MSCs [53].
Many studies suggest that WJ-MSCs tend to be superior to BM-MSCs. They are able to efficiently differentiate toward
functional cardiomyocytes and form myotube structures capable of spontaneous contractions in vitro after coculture with
fetal MSCs but not with adult MSCs (like BM-MSCs) [51]. UC perivascular stem cells exceeded BM-MSCs in cardiac
differentiation potential and beneficial influence on infarcted heart [46]. This phenomenon suggests that “young” (peri-
natal) stem cells possess more profound differentiation potential and as such can be a more powerful tool for regenerative
cell therapy than “old” (adult) stem cells.
Cell transplantations in small animal models (mice and rats) are vital for deciphering mechanisms of MSC actions in
cardiac regenerations. However, experiments on large animal models (such as dogs and pigs) are crucial for closing the gap
between research and clinical utility, as their size, anatomy, and physiology are similar to human.
WJ-MSCs were proved to be efficient in a mini-swine acute myocardial infarction model [54]. Transplanted cells
significantly improved myocardial perfusion and function of the infarcted area in the left ventricle. WJ-MSCs efficiently
engrafted and differentiated into cardiomyocytes and vascular endothelium in 6 weeks after transplantation. They also
284 SECTION j III Umbilical CordeDerived Cells

5. increased vascular density, viable myocardium size, and reduced apoptosis. It is also noteworthy that transplanted
WJ-MSCs promoted recruitment and differentiation of resident cardiac stem cells, proving the important role of cellecell
communication in MSCs regenerative mechanisms (Fig. 21.2).
Another approach for potential future cell therapy of CVDs is tissue engineering. Stem cells isolated from different
regions of UC can be seeded on biocompatible 3D scaffolds for expansion, differentiation, and generation of “patches”
placed in/on damaged tissues [55]. An example of such an approach is artificially engineered pulmonary valves created by
seeding endothelial cells onto synthetic biodegradable scaffold consisting of polyglycolic acid fibers [56]. WJ-MSCs can
be used to create a myocardial patch, which can be helpful in regeneration of infarcted muscle [57]. Biodegradable
macroporous tubes, which allow transport of growth media into the construct, were populated with WJ-MSCs, which
expressed high viability, uniform cell distribution, and alignment due to nutrient provision within bioreactor [57]. Cells
seeded on similar scaffolds can be differentiated toward cardiomyocytes in vitro. Such 3D constructs have a potential to
mimic the structure of the native myocardium and can be used to “patch” a damaged region in infarcted heart.
Although results in preclinical studies are very promising, showing improvement in widely defined cardiac function
(increased ejection fraction, decrease in scar tissue, reversed remodeling, improved contractility, reduced apoptosis,
augmented heart perfusion, and blood vessel density), long-term assessment of WJ-MSCs’ safety and efficacy is still
needed.
WJ-MSCS IN CLINICAL TRIALS
Easily achievable (e.g., off-the-shelf) and highly effective cell type for cell replacement therapy is yet to be determined. For
instance, the embryonic stem cells or induced pluripotent stem cells are also usable sources of cells for regenerative
medicine. Use of these cells, however, is associated with several ethical and technical problems. Another way to obtain
stem cells for regenerative medicine is their isolation from somatic tissues.
FIGURE 21.2 Mechanism of action of mesenchymal stem cells in cardiovascular diseases. Modified from Servier Medical Art.
Wharton’s Jelly MSC and Cardiovascular Damage Chapter | 21 285

6. The key characteristics of WJ-MSCs are their multipotent properties - laying somewhere between pluripotent
embryonic stem cells and unipotent adult stem cells [14]. WJ-MSCs have higher proliferation rates and self-renewal
capacity compared with adult stem cells [9]. The overall evidence indicates that under appropriate vigilance, including
cytogenetic screening [58] such as implemented in the CIRCULATE project, human WJ-MSCs can be safely introduced
in clinical use.
In essence, human WJ-MSCs are allogeneic but nonimmunogenic, when transplanted into another human. Wharton’s
Jelly is also a very promising source of MSCs for clinical application in acute myocardial infarction. WJ-MSCs have been
shown to be safe and beneficial in two independent studies [59,60], with a suggestion of the beneficial effect on infarct size
and left ventricular contractility [60].
With Ethics Committee approval, we have demonstrated safety and feasibility of WJ-MSCs application to treat human
myocardium [59]. In addition, using state-of-the-art imaging techniques [61], we have demonstrated that myocardial
ischemic tissue uptake of WJ-MSCs is 5- to 6-fold greater than that of CD34 cells [62].
In the second study, Gao and coworkers performed double-blinded, randomized controlled multicenter trials.
They showed safety and feasibility of this approach. Monitoring of patients during the 18-month follow-up
demonstrated increase in the myocardial viability and left ventricular ejection fraction (LVEF) in the group transplanted
with WJ-MSCs [60].
Based on the already demonstrated regenerative potential of MSCs, encouraging data from published preclinical
studies, and clinical safety and feasibility work, we propose a novel approach for the ischemic tissue regeneration of
important civilization diseases, including acute myocardial infarction, chronic ischemic myocardial injury, and lower limb
ischemia d an approach based on WJ-MSC allogeneic transplantation.
This novel approach is based on obtaining MSCs from an “unlimited” source, noninvasive and low cost (in comparison
with iPS cells or expansion of autologous bone marrow cells) d Wharton’s Jelly d creating an allogeneic cell bank based
on which an “off-the-shelf” cell therapy product which could be used immediately, providing sufficient number of the cells
for the treatment of a variety of diseases. This treatment strategy, using human WJ-MSCs as a tool to regenerate or
stimulate the regeneration of the damaged tissues, will not only lead to life saving treatments but also enhance their quality
of life. Thus, in the long run, the proposed approach not only will increase the effectiveness of medical treatment (acute and
chronic myocardial disorders) but also may provide the first effective treatment (e.g., in no-option limbs ischemia). This
would bring a new era in the treatment modalities and, in consequence, enormous savings in spending on the hospitali-
zation and social benefitebased care.
CONCLUSIONS
WJ-MSCs possess a number of features, which make them an ideal candidate for future regenerative therapy in CVDs.
Their immunoprivilege ensures histocompatibility and crosses transplant rejection risk out of the equation. As they are not
tumorigenic, their safety is considerably higher than pluripotent cells, which, even after differentiation, remain as a “ticking
bomb” within the patient’s body. WJ-MSCs’ secretion potential guarantees their strong pleiotropic effect on infarcted
heart, not only in resolution of inflammation in infarcted heart, but also in preparation of injured niche for cell homing.
Because they are capable of specific homing into injured sites, their application can be noninvasive (e.g., intravenous). As
WJ-MSCs are capable of cardiac and endothelial differentiation, they are a promising source of cells, which are essential
for repairing infarcted heart. Furthermore, their acquisition (unlike liposuction or bone marrow aspiration) is pain free and
generates abundant perinatal cells. Their ease of allogeneic use makes them an ideal candidate for off-the-shelf products for
personalized therapy in CVDs immediately after infarction (often not feasible in case of autologous cells), time which
sometimes can be life saving. Our group works on preparation of WJ-MSCs as an allogeneic medical agent in the
CIRCULATE project for both preclinical and clinical trials [59]. This approach can close the gap of unmet needs
concerning MSC clinical applications which involves, but are not restricted to, poor availability of abundant autologous
cells in a short period of time after heart failure. It is estimated that approximately 109
cells die in heart attack, which is the
order of magnitude necessary for transplantation within days after heart incident. Allogeneic “off-the-shelf” approaches
appears to be more feasible than autologous cell expansion.
Moreover, because cardiovascular disorders concern mainly elderly people with co-morbidities (e.g., diabetes), it is safe
to assume that their autologous cells would also suffer “co-morbidities,” which can diminish long-term therapeutic effect of
transplanted autologous cells. This risk is overcome by application of “young and healthy” allogeneic cells.
286 SECTION j III Umbilical CordeDerived Cells

7. WJ-MSCs’ features make them extremely promising cells for future regenerative therapy in cardiovascular disorders.
However, there is still a demand for large comprehensive, randomized, controlled trials that establishes crucial features of
MSC in CVDs (e.g., number of cells, time and method of application). Nevertheless, the future for perinatal cells in
regenerative medicine is bright.
ACKNOWLEDGMENT
This work was supported by research grant (Strategmed2/ 265761/10/NCBR/2015) from the National Center for Research and Development.
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