The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization, with Christian Drapeau

Christian Drapeau

View All Contributions By:  

Job Title / Position:

Website:

Contact Information:


The Therapeutic Potential of Stimulating
Endogenous Stem Cell Mobilization
Christian Drapeau1, George Eufemio2, Paola Mazzoni1,
Gerhard D. Roth3 and Susan Strandberg4
1Stemtech International, San Clemente, CA,
2Cardinal Santos Medical Center, Medical Arts Building, Manila,
3Neurology and Psychiatry Center, Ostfildern,
4Stem Cell Center, Spokane, WA
1,4USA
2Philippines
3 Germany
1. Introduction
The past decade has seen a fast and extensive development of various therapies and
treatment protocols based on Adult Stem Cells (ASC) and their application to various
diseases. While some of these treatment protocols have been well documented in the
scientific literature and used in well controlled clinical set ups, others have been developed
and are being used by a growing numbers of clinics throughout the world, without
thorough documentation though nevertheless with good clinical care and with the reports
of very compelling results.
Despite the wide variety of methods, the general procedure guiding these various protocols
follows a series of common steps. The first step is the isolation of stem cells from a source.
For the purpose of banking or clinical application, stem cells can be isolated from a variety
of sources including umbilical cord (Can and Balci, 2011; Zhang et al., 2011), adipose tissuederived
stem cells (Insausti et al., 2011; Zachar et al., 2011), peripheral blood stem cells
(Kolbe et al., 2010; Hofmann et al., 2009), amniotic and placental stem cells (Klein and Fauza,
2011; Tsagias et al., 2011), dental pulp stem cells (Gronthos et al., 2011; Tirino et al., 2011),
olfactory stem cells (Chen et al., 2006; Viktorov et al., 2008), and even human limbal
epithelial stem cells (Vasania et al., 2011).
The second step is proliferation. This is not a necessary step with regard to stem cell
function, however the small number of stem cells present in one umbilical cord, one
placenta, one blood sample, one liposuction or one dental pulp makes clinical application
difficult without the ability to expand the harvested stem cells. Methods to expand
embryonic stem cells have been developed more than a decade ago, however it is only a few
years ago that methods to significantly expand ASC have been developed, leading to an
expansion of the stem cell banking market and greater clinical application (Ivanovic et al.,
2011; Dos Santos et al., 2011; Pineault et al., 2011).
Tissue Regeneration – From Basic Biology to Clinical Application
168
The third step is pre-conditioning or treatment to trigger commitment of the stem cells into a
specific cellular lineage. For example, stem cells can be led to differentiate into dopamineproducing
neuron by an exposure to a cocktail containing sonic hedgehog (SHH), fibroblast
growth factor 8 (FGF8), and basic fibroblast growth factor (bFGF) (Trzaska and Rameshwar,
2011; Wang et al., 2011) or into neurons responding to multiple neurotransmitters by a
simple exposure to retinoic acid and other growth factors (Greco et al., 2008). Likewise, stem
cells can be guided to differentiate in cardiomyocytes by exposure to a cocktail containing
transforming growth factor-beta(1), bone morphogenetic protein-4, activin A, vascular
endothelial growth factor (VEGF), insulin-like growth factor-1, fibroblast growth factor-2,
Epidermal growth factor (EGF), and interleukin-6 (Behfar et al., 2010; Behfar et al., 2008).
Pre-conditioning with these cytokines can also enhance the formation of gap junction and
improve therapeutic efficacy (Hahn et al., 2008). Exposure of stem cells to a cocktail
containing insulin, transferrin, selenium and the GLP-1 (glucagon-like peptide-1) analogue
exendin-4 leads to the formation of insulin-producing pancreatic cells (Docherty, 2009;
Chandra et al., 2009). Various cocktails have been shown to trigger the differentiation of
mesenchymal stem cells in into a wide variety of cell types (Snykers et al., 2011; Arufe et al.,
2009; Keilhoff et al., 2006). Nevertheless, this pre-conditioning step is not essential since ASC
will naturally differentiate into the cell type with which they find themselves, upon contact
with cellular debris or cell marker specific to that cell type. For example, as they migrate into
the heart, stem cells can be triggered to differentiate into cardiomyocytes (Orlic et al., 2001),
or into keratinocytes and skin appendages, insulin-producing pancreatic cells or hepatocytes
as they respectively migrate in a skin wound (Zhang and Fu, 2008), the pancreas (Hasegawa
et al., 2007) or the liver (Theise et al., 2000).
The third and final step is the injection of stem cells into the target organ, in the main artery
leading to the target organ or in the bloodstream from where a number of them will migrate
on their own to the affected organ. In the case of a heart attack for example, stem cells can be
injected in coronary artery (Wollert et al., 2004) or directly in the border zone of the infarct
(Stamm et al., 2003; Tse et al., 2003). Treatment efficacy can vary significantly with the
various methods of injection. For the treatment of acute myocardial infarction, injection in
the border zone of the infarct seems to yield the best results, followed by intracoronary and
intravenous injection, respectively (Karra and Wu, 2008). For the treatment of spinal cord
injury, injection directly in the lesion or in the cerebrospinal fluid seems far superior to
intravenous injection (Lima et al., 2010), yet very compelling cases have been documented
following intravenous stem cell injection or simple bone marrow stem cell mobilization (see
Section 4). For the treatment of diabetes however, intravenous injection seems to yield better
results than stem cells transplantation directly into the pancreas (Hasegawa et al., 2007).
In this multistep procedure in which each step can be accomplished according to a wide
variety of protocols and methods, it remains that peripheral blood stem cells (PBSC) can,
without expansion, pre-conditioning or injection reach various target organs and participate
to the process of tissue repair. This observation has led a number of researchers to look at
the therapeutic potential of simply stimulating Endogenous Bone Marrow Stem Cell
Mobilization (ESCM). This chapter will look in detail into the clinical and therapeutic
potential of ESCM by describing its physiological basis, by reviewing the existing literature
on the clinical application of ESCM and by presenting a few clinical cases.
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization

 
2. The repair system of the body
For ESCM to have any clinical relevance, the demonstration must be made that one of the
natural roles of stem cells in the body is to participate to tissue repair in cases of injury or
degenerative diseases. Therefore, the clinical relevance of mobilizing endogenous bone
marrow-derived stem cells (BMSC) would be to increase the number of circulating stem
cells available to migrate into affected tissues and contribute to tissue repair. For this
phenomenon to be natural: 1) the body must have a mechanism that triggers BMSC
mobilization after an injury; 2) BMSC must traffic in the blood and be recruited by the
injured tissue; 3) in the injured tissue BMSC must proliferate and 4) a mechanism must
exist to trigger the differentiation of BMSC into cells of that tissue.
2.1 Signaling for mobilization
The most common compound known to naturally stimulate BMSC mobilization is
Granulocyte-Colony Stimulating Factor (G-CSF). Discovered in 1985 by Welte et al., G-CSF
is a cytokine secreted by various tissues that stimulates the proliferation, differentiation and
function of neutrophil precursors and mature neutrophils. But G-CSF was also shown to
stimulate BMSC mobilization (Petit et al., 2003; Cottler-Fox et al., 2003), making it a common
tool in protocols of stem cell apheresis for the purpose of cryopreservation and stem cell
transplant (Gordon et al., 2008; Croop et al., 2001).
Given the vital importance of the heart and the fact that cardiovascular diseases are a
leading cause of death in the world, much of the clinical stem cell research has focused its
efforts on the role of stem cells in cardiac repair taking place after acute myocardial
infarction (AMI). A number of studies have revealed the sequence of events taking place
after AMI. A few hours after AMI, the cardiac tissue releases or causes to release G-CSF
(Leone et al., 2006). As its concentration slowly increases in the bloodstream, G-CSF triggers
the release of stem cells from the bone marrow, increasing the number of PBSC which peaks
at around 4-7 days after AMI (Shintani et al., 2001; Leone et al., 2006). It is worth mentioning
that the serum level of G-CSF and the number of PBSC are also increased in cases of chronic
angina (Leone et al., 2006). Similar stem cell mobilization and increase in PBSC have been
documented following skeletal muscle injury (Stout et al., 2007).
Other chemokines such as interleukine-8 (IL-8), Stromal-Derived Factor-1 (SDF-1), Stem Cell
Factor (SCF), Groß, and vascular endothelial factor (VEGF) have been shown to trigger
BMSC mobilization (King et al., 2001; Lapidot & Petit, 2002; Fukuda et al., 2007; Lapid et al.,
2009). Contrary to G-CSF and SCF, which lead to a slow increase in the number of PBSC
over a period of a few days, other cytokines such as IL-8 lead to a significant increase in the
number of PBSC within hours (Fibbe et al., 1999).
As has been described with the heart following AMI, a stroke also triggers the release of
cytokines that induce the mobilization of BMSC and their migration into the brain. For
example, the number of PBSC in stroke patients nearly tripled within 7 days after the stroke
(Hennemann et al., 2008; Paczkowska et al., 2005). In one study, the magnitude of stem cell
release was actually correlated with the functional recovery of the patients (Dunac et al,
2007). Interestingly, the number of circulating stem cells did not increase in patients who
received thrombolysis therapy immediately after their stroke. Therefore, it appears that it is
Tissue Regeneration – From Basic Biology to Clinical Application
170
the lingering injury that slowly leads to the mobilization of stem cells from the bone
marrow.

 
Finally, injuries to the skin and bones were also shown to trigger mobilization of BMSC and
their migration into the injured tissue. For example, within 24 hours of a severe burn, a
rapid increase of up to 9-fold in the number of PBSC has been observed in the blood of burn
patients (Fox et al., 2008). Furthermore, the size of the area of the body affected by the burns
strongly correlated with the magnitude of the mobilization. The affected skin also released
cytokines such as SDF-1 and VEGF, which are involved in the migration of PBSC to the skin
and their differentiation into blood vessels, respectively (Mansilla et al., 2006). In one study
(Lee et al., 2008), the number of PBSC peaked around 3 days after a bone fracture and
rapidly returned to basal level within a few days. These results were confirmed in another
study in which stem cells were shown to migrate to the fracture site and to promote
neovascularization. The formation of new vessels was shown to peak at the fracture site 7
days after the fracture, which corresponds to the early phase of ossification of the fracture
line (Matsumoto et al., 2008). Therefore BMSC mobilization was documented to naturally
follow an injury or even be associated with chronic conditions.
The natural process by which stem cells are mobilized from the bone marrow is still not
fully understood. Contrary to most tissues in which SDF-1 is secreted consequent to an
injury or a degenerative condition, in the bone marrow SDF-1 is constitutively produced
and released, and binding of SDF-1 to its exclusive receptor CXCR4 leads to the
externalization of adhesion molecules, namely integrins, which allow for the adherence of
stem cells to the bone marrow matrix. The binding of SDF-1 to CXCR4 is referred to as the
SDF-1/CXCR4 axis. The general understanding is that disruption of the SDF-1/CXCR4 axis
reduces the expression of adhesion molecules, leading to a reduction in the adherence of
stem cells to the bone marrow matrix and the consequent mobilization of stem cells. Various
compounds known to trigger stem cell mobilization all affect the SDF-1/CXCR4 axis in
various ways.
For example, G-CSF disrupts the SDF-1/CXCR4 axis by activating a series of proteolytic
enzymes including elastase, cathepsin G, and various matrix metalloproteinases (MMP2 and
MMP9) that inactivate SDF-1 (Mannello et al., 2006; Jin et al., 2006; Carion et al., 2003).
AMD3100 is a newly developed BMSC mobilizer and it acts by blocking CXCR4, disrupting
the SDF-1/CXCR4 axis (Broxmeyer et al., 2005). A blocker of L-selectin was recently isolated
from the cyanophyta Aphanizomenon flos-aquae and shown to trigger BMSC mobilization
(Jensen et al., 2007). Inhibition of L-selectin leads to a down-regulation of CXCR4 expression,
partially disrupting the SDF-1/CXCR4 axis. The mobilization mechanism of IL-8, SCF,
VEGF and Groß are not well understood.
Therefore, the human body has a mechanism to naturally mobilize BMSC which can then
traffic to various areas of the body and contribute to tissue regeneration and repair.
2.2 Extravasation & recruitment
Recruitment is the process by which PBSC are recruited by a specific tissue signaling for
repair. The process of PBSC recruitment in a tissue takes place predominantly at the level
the postcapillary venule, in a manner similar to neutrophils (Henschler et al., 2008).
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
171
In brief, the sudden drop in blood pressure taking place at the postcapillary venule triggers
turbulence whose shear force mechanically activates L-selectin which in turn triggers the
externalization of CXCR4, making PBSC more responsive to signals coming from tissues. If
the tissue is in need of repair, it is secreting SDF-1 as well as other compounds such as stem
cell factor (SCF) and hepatocyte growth factor (HGH) (Kucia et al., 2004; , Neuss et al., 2004)
that diffuse locally to the capillaries. Binding of SDF-1 and SCF to their specific receptors
(e.g. CXCR4 and c-Kit) leads to the expression of adhesion molecules at the surface of stem
cells (Voermans et al., 2000; Peled et al., 1999; Peled et al., 2000). Through a complex
interaction of microvilli at the surface of both the capillary and PBSC, the stem cells initiate
the process of tethering and then arrest on the capillary endothelium (Middleton et al., 1997;
2002).
Following firm attachment, SDF-1 and HGH continue to bind to their respective receptors
CXCR4 and c-met at the surface of SC, which triggers the release and activation of matrix
metalloproteinases (MMPs) which digest the endothelial lining, allowing for the
extravasation of PBSC (Mannello et al., 2006; Janowska-Wieczorek et al., 2000; Neuss et al.,
2004; Ries et al., 2007).
When studying the migration behavior of SCs to a wide array of chemokines, SCs were
found to migrate only toward SDF-1 (Wright et al., 2002; Jo et al., 2000). The migration to
SDF-1 is a polarized phenomenon toward the chemokine source (chemotactic) and not a
simple random motion (chemokinetic), as SCs only migrated in a gradient of SDF-1 and not
when SDF-1 is uniformly distributed in the media.
SDF-1 is normally secreted, to some extent, by cardiomyocytes (Askari et al., 2003), skeletal
muscles (Ratajczak et al., 2003), liver (Hatch et al., 2002; Kollet et al., 2003), brain (Bagri et al.,
2002; Lazarini et al., 2003; Zou et al., 1998), and kidney (Schrader et al., 2002). However, its
secretion increases during tissue damage such as AMI (Wojakowski et al., 2004; Abbott et al.,
2004), ischemia (Takahashi et al., 1999, Iwaguro et al., 2002), toxic liver damage (Kollet et al.,
2003; Swenson et al., 2008; Hatch et al., 2002), and excessive bleeding (Ratajczak et al., 2004).
In the tissue, the process of migration toward the site of injury relies on the interaction
between CD44 and its ligand hyaluronic acid (HYA). HYA is one of a family of
polysaccharides known as glycosaminoglycans (GAGS), typically found in the connective
tissues of vertebrates. Alongside other proteins such as collagen, elastin, fibrillin, fibronectin
and laminin, GAGS and HYA constitute the extracellular matrix (ECM) of most tissues.
Studies in rats have shown that half of the HYA found in the body is in the skin, while 25%
is found in joints and bones together (Reed et al., 1988). The rest is distributed somewhat
equally in muscles and viscera (Clarris and Fraser,1968; Comper and Laurent, 1978) where
the highest concentrations are found in connective tissue that form the ECM of most tissues.
After extravasation, CXCR4 on the surface of SC and SDF-1 secreted by the effected tissue
continue their interaction which, in the tissue, leads to the formation of pseudopodia (see
Figure 1) toward the source of SDF-1 secretion. As SDF-1 binds to CXCR4 in the tissue,
CD44 adhesion molecules are externalized at the tip of the pseudopodia, leading to
adhesion to HYA pathways within the tissue. In the tissue, the binding of CD44 to HYA is
transient, as CD44 molecules shed soon after binding to HYA (Friedl et al., 1995), thus
enabling pseudopodia detachment from the ECM. CD44 can also be cleaved by specific
enzymes whose secretion is enhanced by SDF-1 (Heissig et al., 2002; Okamoto et al., 1999;
Tissue Regeneration – From Basic Biology to Clinical Application
172
Janowska-Wieczorek et al., 1999). Through this process, stem cells can migrate within the
tissue toward the site of injury.
The fact that PBSC primarily migrate to organs affected by an injury or a degenerative
process has been documented in several studies. For example, in the cases of male recipients
of liver transplants from female donors, biopsies performed 4-13 months after
transplantation contained a significant number of Y-chromosome positive hepatocytes (16%
to 43%). In one patient who suffered from hepatitis C after liver transplant and died 4.5
months after transplantation, up to 43% of the transplanted liver was made of Ychromosome
positive hepatocytes in comparison with 5% after 4.5 months in a woman with
a sound liver who received a bone marrow transplant from a man (Theise et al., 2000).
Investigations carried out on archival autopsy and biopsy liver specimens obtained from
two women who received a bone marrow transplant from male donors for the treatment of
leukemia revealed that 5 to 10% of the liver had been replaced by donor derived BMSCs
after 4.5 and 13 months, respectively (Theise et al., 2000).
Fig. 1. CD44 is localized to the leading edge of polarized human stem cells migrating
toward SDF-1. Cord blood-derived CD34+ cells were plated on HA coverslips and allowed
to adhere for 30 minutes before recording cell movement. The position of SDF-1 source is
indicated by arrowheads. (A-C) Phase contrast microscopy of untreated cells (A), cells
stimulated with polarized source of SDF-1 (B), and cells treated with anti-CD44 mAb F10-44-
2 and stimulated with polarized source of SDF-1 (C). (D-F) Cells treated as above were fixed
5 and 30 minutes after exposure to polarized source of SDF-1 and indirectly immunolabeled
with antihuman CD44 mAb (red) and anti-CXCR4 mAb (green). An arrow is pointing to the
fine CD44-positive protrusions at the direction of SDF-1. Bars = 5μm.
(Taken from Avigdor et al., 2004)
Similar observations were made on men who received cardiac transplants from female
donors. Analyses of tissue samples from biopsies revealed that an average of 0.1% (Muller et
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
173
al., 2002) to 15% of cardiomyocytes were Y-chromosome positive (Quaini et al., 2002;
Laflamme et al., 2002). In one patient who died of cardiac rejection, 29% of the
cardiomyocytes contained the Y chromosome in ‘‘hot spots’’ of high cardiac repair. In male
patients who received lung transplants from female donors, recipient-derived BMSCs cells
were detected as bronchial epithelial cells, type II pneumocytes, and seromucous glands in
the transplanted lungs of all tested patients. In patients suffering from chronic damage, up
to 24% of bronchial epithelial cells carried the Y-chromosome, indicating ongoing repair of
damaged tissue by the recipient’s own stem cells (Kleeberger et al., 2003).
In one study investigating this process of tissue repair by BMSC, irradiated mice were
transplanted with GFP-positive SC before being injected in the right tibialis muscle with a
large dose of cardiotoxin, which led to a loss of mobility within a few days. Yet, after eight
weeks the injured muscle showed massive regeneration, with the right tibialis muscle
significantly reconstructed with GFP-positive myocytes. By contrast, the contralateral noninjured
leg showed very small incorporation of GFP-positive myocytes (Drapeau et al.,
2010). Similar observations were made by Abedi et al. (2004), using smaller injections of
cardiotoxin. Four weeks after the injection of cardiotoxin in the right leg, the area of the
injection contained 1-2% of GFP-positive muscle cells while the left leg showed no
fluorescence at all.
Similar observations were made in models of skin injury (Abedi et al., 2004). In a similar
protocol, irradiated mice were transplanted with GFP-positive stem cells. BMSCs were then
mobilized during five days using G-CSF. On the fourth day, mice were subjected to punch
biopsies on their flank. The area of the injury was rebiopsied and sutured 48 hours and 1
month after the injury, in order to assess the incorporation of GFP-positive cells in the
healing skin. While analysis at 48 hours showed significant infiltration by GFP-positive
undifferentiated stem cells in the deep layer of the skin (hypodermis), after 4 weeks there
was a large number of GFP-positive tissue cells in the dermis composing the structure of the
healed skin, such as keratinocytes, sebaceous glands, blood vessels, and some rare muscle
fibers and hair follicles. In the control animals that received G-CSF but no punch biopsy,
none of these skin structures were positive for GFP, indicating that the few SC that had
migrated in the skin had done so randomly.
In other studies looking at the incorporation of BMSCs in injured tissues, directed migration
was demonstrated in the gut after section of an intestinal segment, (Hayakawa et al., 2003)
in the heart after AMI (Orlic et al., 2001a; Fukuhara et al., 2004) or induced cardiomyopathy,
(Hisashi et al., 2004) in the brain after stroke, (Sanchez-Ramos et al., 2002; Hoehn et al., 2002)
and in the liver after drug-induced liver damage (Abedi et al., 2004). Taken altogether these
studies clearly establish that BMSCs primarily migrate to areas subjected to injury, damage
or simple degeneration.
2.3 Proliferation
When stem cells reach the site of an injury they must proliferate and expand, as there are not
enough PBSC to accomplish full repair of any significant injury or degenerative process.
Several chemokines such as SDF-1 have been reported to enhance stem cell proliferation
(Bonavia et al., 2003). The direct effect of SDF-1 on cell proliferation and survival is not well
understood, but SDF-1 has been found to stimulate the proliferation and survival of stem

Tissue Regeneration – From Basic Biology to Clinical Application
174
cells under certain experimental conditions (Broxmeyer et al., 2003). In tissues, SDF-1
appears to act as a “cellular survival factor” (Hwang et al., 2006). Insulin-like growth factor
(IGF-1), when coupled with epidermal growth factor (EGF) or fibroblast growth factor-2
(FGF-2), was shown to support the proliferation and survival of neural (Arsenijevic et al.,
2001) and muscle stem cells (Deasy et al., 2002). Extracellular nucleotides were also shown to
support the proliferation of brain stem cell (Mishra et al., 2006).
2.4 Differentiation
The ability of adult stem cells to differentiate into various cell types has been well
documented, though the mechanism behind such transformation is still not well
understood.
As reported by Krause et al. (2001), 11 months after injection of male stem cells in female
mice, Y-chromosome bearing cells were found in various tissues including the liver, muscle,
skin, lung, and intestine. It has been well demonstrated that BMSC can differentiate into a
wide variety of cell types including myocytes (Ferrari et al, 1998), hepatocytes (Lee et al.,
2004), epithelial cells (Krause et al., 2001), neurons (Mezey et al., 2003; Sanchez-Rarnos et al.,
2000; Woodbury et al., 2000), retinal cells (Tomita et al., 2002), endothelial cells and
cardiomyocytes (Jackson et al., 2001; Orlic et al, 2001), gastrointestinal epithelium (Krause et
al., 2001; Okamoto et al., 2002), pancreatic endocrine cells (Ianus et al., 2003), bone and
cartilage (Pereira et al., 1995; 1998).
Although little work has been done in this field and many questions remain to be answered,
two possible mechanisms have been proposed for SC differentiation.
One proposed mechanism is cellular fusion, which takes place when two cells fuse together
to become one cell. A few studies have suggested that SC have the ability to fuse with
somatic cells, rescuing the target cell (Tarada et al., 2002; Vassilopoulos et al., 2003; Spees et
al., 2003). Although there is clear evidence that this phenomenon did take place in a few
experiments and that it may take place naturally in certain tissue such as the heart (Nygren
et al., 2004), it is unlikely to be a significant physiological phenomenon (Wurmser and Gage,
2002). For example, while the process of fusion involves the interaction of one single SC with
one somatic cell, therefore a ratio of 1:1, the extent of SC-mediated tissue repair that has
been documented in numerous studies, involving various tissues, far exceeds the actual
number of SCs migrating in the tissue. Furthermore, the process of cellular fusion results in
a cell that is tetraploid. Although this has been observed in a few in vitro studies, it has been
a very rare observation in vivo. In fact, even in vitro, relatively harsh conditions had to be
used in order to obtain cellular fusion. Finally, cellular fusion would imply the merging of
two different cellular membranes, a process that in itself is rigged with challenges, as cells
are designed not to fuse.
So, although cellular fusion could possibly naturally take place in the body, it is unlikely to
contribute significantly to the process of repair that has been documented with ASCs. The
other most likely mechanism is differentiation through contact with cellular components
when the affected tissue is locally subjected to the action of various matrix
metalloproteinases (MMPs) or differentiation induced by cytokines released by neighboring
cells.
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
175
This process was beautifully put in evidence by the study of Jang et al. (2004) where
hematopoietic stem cells (HSC) were co-cultured with either normal or damaged liver tissue
separated by a semi-permeable membrane with 0.4 μm pores. Using immunofluorescence to
detect markers specific to either HSC (CD45) or hepatocytes (albumin), the authors could
follow the transformation of the HSC population. When HSCs were cultured alone for 8
hours, they only expressed CD45 and no albumin. However, when HSCs were exposed to
injured liver tissue, they rapidly became positive for albumin. Over time, the population of
cells positive for CD45 began to decrease as the population positive for albumin began to
increase. Albumin-positive cells were seen as early as 8 hours and constituted 3.0% of the
cell population at 48 hours. The conversion was minimal and delayed when HSC were
exposed to undamaged liver (control for injury). Therefore the presence of an injury appears
to be an important factor in the process of SC differentiation into a specific type of somatic
cells.
The authors further investigated the phenomenon of differentiation by tracking the presence
of various markers found in developing fetal liver cells, such as αFP, and in mature
hepatocytes, such as CK18, albumin, fibrinogen, and transferrin. They found that αFP was
expressed only at 8 hours and was lost thereafter. On the other hand, the expression of
CK18, albumin, fibrinogen and transferrin each increased over time. While at time 0 the
HSCs expressed only CD45, after as little as 8 hours all liver-specific markers were positive.
So during the differentiation process, the SC seemed to take a route similar to the
development of hepatocytes in the developing fetus, leading to mature hepatocytes within
less than 48 hours. This retracing of fetal development has also being documented in
cardiopoiesis (Behfar et al., 2008).
In this study, differentiation did not involve cellular fusion, as the new liver cells only
contained one set of chromosomes. The differentiation was necessarily triggered by
signaling molecules produced by the damaged tissue. It has been suggested that MMPs
produced by damaged tissue could be playing an important role in SC differentiation by
digesting specific ECM components that would then diffuse and get into contact with SCs.
Binding of such compounds to specific receptors would activate internal messengers that
would trigger the process of differentiation by activating specific genes, as suggested by the
high level of mRNA found in differentiating cells. (Mannello et al., 2006; Chavey et al., 2003)
Gap junction intercellular communication (GJIC) and tunneling nanotubules (TNTs) could
constitute other mechanisms playing a significant role in SC differentiation, by direct cell-tocell
contact (Behfar et al., 2010). The diameter of one gap junction is around 2 nm and the
molecular size cut-off level is around 1-2 kDa, which is sufficient for the intercellular
exchange of ions, nucleotides and even small proteins (Neijssen et al., 2005). GJIC is known
to play a crucial role in modulating several cellular functions, to the point that impaired or
lack of GJIC has been associated with severe diseases (Dasgupta et al., 1999). It has been
suggested that GJIC could play an important role in SC differentiation (Loewenstein and
Rose, 1992). Likewise, recent studies indicated that two cells can also exchange information
via TNTs (Rustom et al., 2004). TNTs would form a cytoplasma bridge between two cells
that could be large enough to allow the transport of large molecules or even whole cell
organelles. Such information could play a role in the finalizing process of differentiation,
though much work needs to be done in this field before we obtain a better understanding of
the role of GJIC and TNTs in stem cell differentiation.
Tissue Regeneration – From Basic Biology to Clinical Application
176
2.5 Paracrine effect
Finally, a number of studies investigating the effect of BMSC on various diagnostic entities
have revealed that oftentimes the extent of the benefits observed cannot be accounted for by
the sheer number of BMSC that have differentiated into somatic cells, even when there is
clear evidence of tissue regeneration mediated by newly formed cells. For example,
irradiated female mice transplanted with transgenic GFP+ BMSC showed much better
recovery from experimental stroke after G-CSF-induced BMSC mobilization when
compared to non-mobilized control mice (Kawada et al., 2006). Both motor and cognitive
functions were improved by BMSC mobilization, and the treatment also reduced infarct
size. However, using bromodeoxyuridine (BrdU), it was observed that a significant number
of the newly formed brain cells did not derive from BMSC but rather from local neural stem
cells. Similar results were seen with spinal cord lesion and muscle injury (Kinnaird et al.,
2004; Osada et al., 2010). The regenerative effect is believed to be triggered by the secretion
of growth factors and paracrine signaling by BMSC (Uccelli et al., 2011).
The paracrine effect has been best put in evidence in the heart after AMI. Delivery of BMSC
to ischemic cardiac tissue has led to a significant increase in the concentration of IL-10,
interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and other cytokines in the cardiac
tissue, which contributed to neovascularization and reduction of infarct size (Kamihata et
al., 2001; Burchfield et al., 2008; Gnecchi et al., 2005; Mirotsou et al., 2011). Condition media
in which BMSC were exposed to hypoxia proved to be cytoprotective to cardiomyocytes and
was able to reduce infarct size (Gnecchi et al., 2006).
In all, a growing body of evidence supports the hypothesis that as BMSC migrate into
tissues, aside from differentiating into cells of the target tissue, they also exert their
regenerative effect at least in part -and maybe even to a large extent- through the secretion
of paracrine signaling compounds.
3. ESCM as a treatment approach
Given the fact that SDF-1 is secreted by various organs and tissues upon injury or
degeneration, it would follow that the number of PBSCs should be an important parameter
in the overall effectiveness of SC-mediated tissue repair and regeneration. A higher number
of PBSC would mean more SC available to respond to SDF-1 signaling and migrate into
tissues. In this regard, Tomoda et al. (2003) reported that after a heart attack individuals
with a higher number of PBSC showed greater recovery of cardiac functions after 6 months
when compared to people having fewer PBSC at the time of the cardiac event.
In a prospective study using more than 500 individuals, Werner et al. (2005, 2007) put in
evidence that the number of PBSC is a critical parameter in the role of SCs in tissue repair.
The authors quantified the baseline number of PBSC in 519 individuals (average 66.6±10.8
years old) at risk for cardiovascular problems, and monitored their condition for one year. A
first major cardiovascular event occurred in 214 patients. After adjustment for age, sex,
vascular risk factors, and other relevant variables, increased levels of PBSC were associated
with reduced risk of death from cardiovascular causes, lesser risk of a first major
cardiovascular event, greater revascularization and lesser frequency of hospitalization.
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
177
Similar data has been obtained by Vasa et al. (2001) who documented that a higher number
of endothelial progenitor cells (EPC) was associated with greater cardiovascular health. In
fact, hypoxia has been associated with the secretion of SDF-1 and VEGF by the ischemic
tissue (Schioppa et al., 2003; Bachelder et al., 2002). Circulating EPCs therefore are attracted
to the ischemic heart by the action of SDF-1 and the migrating EPCs contribute to the
formation of new blood vessels upon the action of VEGF (Lee et al., 2007). Therefore, a
higher number of EPCs or PBSC helps maintain optimal blood flow and a strong cardiac
tissue.
The link between disease formation and the number of circulating stem cells is not limited to
the heart. Similar observations have been made with muscular dystrophy where the rate of
progression of the disease has been linked to the number of circulating stem cells and for
which the number of PBSC is now considered one of the most important predictors of the
disease progression (Marchesi et al., 2008). Likewise, the progression of pulmonary arterial
hypertension (Diller et al., 2008; Junhui et al., 2008), arthritis (Herbrig et al., 2006; Grisar et
al., 2005), atherosclerosis (Zhu et al., 2006 ), lupus erythematosus (Westerweel et al., 2007;
Moonen et al., 2007), kidney failure (Choi et al., 2004; Herbrig et al., 2004; Eizawa et al.,
2003), migraine (Lee et al., 2008), erectile dysfunction (Baumhakel et al., 2007) and other
diseases have all been linked to a reduction in the number of PBSC. Recently, a direct
relationship has been established between the number of PBSC and the development of
diabetes, linking impaired fasting glucose, impaired glucose tolerance, and insulindependent
diabetes mellitus to progressively lower levels of PBSCs (Fadini et al., 2010).
If the number of PBSC constitutes such a key parameter in the process of SC-mediated tissue
repair, therefore increasing the number of PBSC could constitute a therapeutic approach.
Following is the description of a number of clinical trials investigating the clinical potential
of ESCM in various diseases or injuries.
3.1 Acute myocardial infarction
The heart has been traditionally seen as having little regenerative capabilities after birth,
although many recent studies have challenged this view. Evidence clearly suggests that
there is a low level of constant regeneration of cardiac cells (Soonpaa and Field, 1998;
Bergmann et al., 2009; Quaini et al., 2002), and the number of dividing cells can increase by
up to 10-fold in chronic heart disease or after AMI (Kajstura et al., 1998; Beltrami et al.,
2001). Yet, this level of proliferation seems insufficient to rescue the cardiac muscle after
AMI (Schwartz and Kornowski, 2003) and survivors of heart attacks are left with reduced
quality of life and little prospect for improvement.
A number of studies in animals using G-CSF and SCF have shown that ESCM can lead to
significant cardiac repair after AMI. Injection of SCF and G-CSF for 8 days after inducing
AMI significantly increased the number of PBSCs, which led to the migration of PBSCs into
the myocardium (Orlic et al., 2001b). Twenty-seven days after AMI, a band of newly formed
cardiac tissue occupied more than 75% of the infarcted region of the ventricule and newly
formed blood vessels were supplying the infarcted tissue. The blood vessels were
surrounded by smooth muscles and microscopic observations revealed the presence of red
blood cells, indicating that the newly formed arterioles integrated structurally with the
remaining functional vasculature. By comparison, in control animals the ventricular wall
Tissue Regeneration – From Basic Biology to Clinical Application
178
was filled with scar tissue covering the entire area of the infarct and no new blood vessels
could be seen.
In summary, while only 17% (9 of 52) of the untreated animals survived AMI, showing
severe signs of cardiomyopathy and compromised blood circulation, up to 73% (11 of 15) of
the animals treated with G-CSF survived with significantly improved cardiac function and
restored blood circulation. After 27 days, ejection fraction was 114% greater in the treated
group and other parameters such as end-diastolic pressure, systolic pressure, and other
parameters of cardiovascular function were all improved in treated versus non-treated mice.
Injection of G-CSF, however, can have significant negative effect in humans if done at large
dose for more than 5-6 days (Bensinger et al., 1996; Shimoda et al., 1993). At lower dose and
with shorter treatment duration, human trials have so far delivered mitigated results,
though the approach remains promising. While some groups did report very promising
results (Ince et al., 2005; Sesti et al., 2005), others reported no effect at all (Ellis et al., 2006;
Zohlnhofer et al., 2007; Ripa and Kastrup, 2008) . A comprehensive review of the various
studies however reveals that each study used slightly different protocols with regard to the
time of treatment after AMI (from hours to 3 months), as well as the intensity and duration
of the treatment, suggesting that ESCM could indeed hold great promise once the most
effective treatment protocol has been developed (Abdel-Latif et al., 2008).
For example, Wojakowski et al. (2006) reported in 43 cardiac patients that if the patients
were treated early after AMI (<12 hours) with G-CSF, the number of PBSCs following BMSC
mobilization correlated with the extent of cardiac repair. In another study, G-CSF was
injected within 5 days post-AMI in 41 patients at high risk for unfavorable left ventricular
remodeling. Five months after G-CSF treatment, ejection fraction had improved 12.5%
compared to no improvement in the control group (Leone et al., 2007). The improvements in
cardiac function appeared to be linked to the prevention of left ventricular remodeling.
A meta-analysis reviewing the effectiveness of BMSC mobilization for the treatment of AMI
included 7 studies and a total of 364 patients. The analysis concluded that treatment with GCSF
can improve LV ejection fraction if the treatment is administered early after the heart
attack (Kang et al., 2007). However, in spite of the improvements in ejection fraction, other
general parameters of cardiovascular health such as ventricular arrhythmia,
rehospitalization for heart failure, and the composite of other cardiovascular events (i.e.,
death from heart attack, recurrent heart attack, and stroke), were not significantly different
in the G-CSF treatment groups compared with the control groups. Similar results were
reported by another meta-analysis that included eight studies and 385 patients (Abdel-Latif
et al., 2008).
So, it remains unclear whether the simple mobilization of BMSCs can constitute an effective
treatment for AMI. While some studies have yielded promising results, others suggest no
benefits at all. However, positive results obtained in some studies should not be denied on
the basis of the negative results obtained in others. Reconciliation of all this data and the
development of an effective treatment protocol will most probably come through the
determination of optimal treatment parameters: 1) intensity of ESCM, 2) duration of the
treatment, 3) time after AMI, 4) number of treatments received over time, and 5) other yet
unidentified parameters. Compounds other than G-CSF might also be discovered that could
provide more consistent results (Broxmeyer et al., 2005; DeClercq, 2005).
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
179
3.2 Stroke
Many studies have shown that extensive neuronal death in the brain after a stroke triggers
the migration of neural stem cells to the site of injury, followed by their proliferation and
differentiation into neurons and glial cells (Peterson, 2002; Fallon et al., 2000; Arvidsson et
al., 2002; Nakatomi et al., 2002; Schmidt and Reymann 2002). However, this natural process
does not appear to be sufficient to produce significant functional recovery (Yamamoto et al.,
2001; Magavi and Macklis, 2002).
As with the heart after AMI, stroke has been associated with BMSC mobilization. Studies
have shown that the number of PBSC in stroke patients can increase up to 3-fold within 7
days after the stroke (Hennemann et al., 2008; Paczkowska et al., 2005). In one study, the
magnitude of BMSC mobilization was correlated with the patients’ functional recovery
(Dunac et al, 2007).
When rats were injected with rat (Chen et al., 2001; Pavlichenko et al., 2008; Willing et al.,
2003) or human SC (Li et al. 2002) after an induced stroke, significant motor and cognitive
improvements were observed. Although a significant number of BM-derived cells could be
identified as newly formed neurons and glial cells in the stroke foci, they accounted for only
a small percentage of the total number of newly formed brain cells. Most of the newly
formed brain cells are believed to be derived from neural SC upon the action of paracrines
secreted by the migrating SC. Similar results were obtained using human umbilical cord
stem cells (HUCSCs) where intravenous injection of HUCSCs 24 hours after a stroke greatly
improved functional recovery (Chen et al., 2001). Injection of HUCSCs 7 days after the
stroke still led to significant functional recovery, though the extent of the recovery was less
than with treatment at 24 hours.
Mobilization of BMSCs induced by G-CSF was shown in a number of studies to improve the
outcome of a stroke. For example, when tested 14 and 28 days after a stroke, animals treated
with G-CSF showed much greater body coordination than control animals (Shyu et al.,
2004). When the brains were analyzed using imaging, the infarcted area was much smaller
in the treated group (61 mm3) when compared to the control group (176 mm3). All these
benefits were greatly reduced when the animals were pre-treated with a blocker of CXCR4,
indicating that the observed effects were dependent upon the migration of BMSCs into the
brain.
Similar results have been reported by other scientific teams (Six et al., 2003; Kawada et al.,
2006). For example, after repopulating the bone marrow with GFP-positive stem cells, GCSF-
induced BMSCs mobilization immediately and roughly 2 weeks after inducing a stroke
dramatically improved motor and cognitive performances four weeks after the stroke, as
measured by using the Morris water maze (Kawada et al., 2006). In this study, while all the
mice in the treated group reached the platform within 40 seconds, none of the control mice
reached the submerged platform within the allotted 120 seconds. As in the study by Shyu et
al. (2004), the infarct size was much smaller in the treated animals when compared to
control. Using BrdU it was observed that the number of new brain cells found in the
infarcted area was much higher in the treated group than in the control group. Yet very few
of the new brain cells were GFP-positive, supporting the view that as they migrate in the
brain BMSCs secrete growth factors that support the proliferation and differentiation of
neural stem cells (Yoo et al., 2008). BMSCs also support neovascularization, which further
Tissue Regeneration – From Basic Biology to Clinical Application
180
contributes to the regeneration of the brain tissue (Lee et al., 2005; Hess et al., 2002; Kan et
al., 2005).
Although much of this work needs to be reproduced in humans, ESCM for the treatment
of stroke appears promising and would constitute a safe approach to the treatment of
stroke.
3.3 Diabetes
The ability of BMSCs to leave the bone marrow, migrate to the pancreas and become insulinproducing
cells was beautifully shown by Ianus et al. (2003). In brief, female mice were
lethally irradiated and then transplanted with male BMSCs that express, using a CRE-LoxP
system, GFP if the insulin gene is actively transcribed. When analyzed 4-6 weeks after the
transplantation, GFP-positive cells were found in the pancreas (Figure 2). The GFP-positive
Fig. 2. FISH and immunofluorescence marking of BM-derived insulin-producing cells.
Immunofluorescence and FISH of isolated, dispersed pancreatic islet cells after
transplantation of lethally irradiated female mice with male BMSCs that express, using a
CRE-LoxP system, GFP if the insulin gene is actively transcribed. a) Bright-field phase, b)
GFP imaging note slight autofluorescence of control isolated islet cells; c) Immunostaining
with rhodamine X–labeled secondary antibody for insulin; d) FISH for Y chromosome (in
yellow) and nucleus stain with DAPI (blue). Y chromosome is present only in GFP-positive
cells. Scale bar, 5 μm; X630. (Taken from Ianus et al., 2003)
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
181
cells were also positive for insulin, for insulin RNA, and for Y-chromosome, demonstrating
that they originated from the transplanted BMSCs. These cells showed functional
characteristics typical of normal pancreatic ß-cells, such as fluctuations of intracellular
calcium upon exposure to various concentrations of glucose. Within the time frame of that
study (4–6 weeks), 1.7–3% of BM–derived GFP-positive cells were detected in the pancreatic
islets. In a similar study, BMSCs were also shown to participate into the development of
new blood vessels, further supporting the regeneration of the pancreatic tissue (Mathews et
al., 2004; Gao et al., 2008).
Then, using a protocol similar to that used by Ianus et al., Hasegawa et al. (2007) further
demonstrated that mobilization of BMSCs was not only effective but essential for pancreatic
regeneration. Hasegawa et al. induced diabetes by injection of streptozotocin (STZ) in
lethally irradiated female mice followed by infusion of BMSC from GFP transgenic mice.
Infusion of BMSCs led to the incorporation of GFP-positive BMSCs into islets of Langerhans
in the pancreatic tissue, partially restoring pancreatic islet number and size, and improving
STZ-induced hyperglycemia. However, when the same experiment was done while simply
infusing the pancreas with BMSCs, without preirradiation, no improvement was obtained.
Furthermore, when the experiment was repeated with full BMSC transplant in a model of
mice with impaired ability to mobilize stem cells, no benefits were obtained. Therefore,
natural mobilization of BMSCs from the bone marrow appears essential for the regeneration
of pancreatic function after inducing diabetes with STZ.
Fig. 3. Pancreatic islets of STZ-treated mice receiving subsequent bone marrow transplant
(BMT). Double immunostaining of pancreases with anti-insulin and anti-glucagon
antibodies. Green indicates insulin-positive and red glucagon-positive cells. Pancreases from
normoglycemic control mouse (a), hyperglycemic control mouse (b), and STZ-treated mouse
receiving BMT (c). BMT improved STZ-induced hyperglycemia. (Taken from Hasegawa et
al., 2007)
In one recent study in humans, ESCM showed great promise in the treatment of diabetes.
The study selected individuals recently diagnosed for diabetes and the treatment consisted
of both stem cell mobilization and autologous stem cell transplant. The patients first
received injections of G-CSF in order to harvest PBSC, followed later by autologous stem cell
thansplant and, 5 days post-transplant, a second round of G-CSF treatment. The endpoints
monitored in the study were overall morbidity along with temporal changes in exogenous
insulin requirements. Before the treatment, all patient required daily insulin injection. By the
end of the study, 14 of the 15 patients had experienced insulin-free episodes ranging
between 1 and 35 months (mean 16.2 months) (Voltarelli et al., 2007).
Tissue Regeneration – From Basic Biology to Clinical Application
182
In this study the patients benefited from two instances of ESCM and one instance of
autologous SC transplant. It is not possible to determine what were the respective
contributions of the ESCM and SC injection, however it is likely that the mobilizations by
themselves significantly contributed to the benefits experienced. While the first mobilization
lasted several days and the second mobilization lasted about one week, there was only one
instance of stem cell injection.
Diabetes is an interesting disease to study the link between disease progression and the
number of circulating PBSCs, as it follows a series of relatively well defined stages with
regard to carbohydrate metabolism status, namely normal glucose tolerance (NGT),
impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and newly diagnosed
diabetes mellitus (DM). Fadini et al. (2010) quantified the number of circulating CD34+ cells
by flow cytometry in 425 individuals divided among these four stages of disease
progression. The data showed a clear trend of decreased number of PBSCs with disease
progression through IFG, IGT and DM (Figure 4). The number of circulating PBSCs was
significantly lower in the IGT and DM groups when compared to the NGT group. The
reduction in the number of PBSCs can either be a consequence of higher blood glucose levels
that might affect the ability of stem cells to mobilize from the bone marrow or a causal factor
in the development of DM whereby a reduced number of circulating PBSCs reduces the
ability of the pancreas to renew itself over the years, or both. This supports the view
previously suggested that diabetes could be a stem cell disease (Fadini et al., 2009).
Fig. 4. Variation of circulating CD34+ cells and diabetes. Variation of circulating CD34+ cells
in patients grouped according to carbohydrate metabolism, namely normal glucose
tolerance (NGT), impaired fasting glucose (IFG), impaired glucose tolerance (IGT) or
diabetes (DM) duration, as appropriate. The mean value of patients with NGT was taken to
represent the zero point. Bars indicate 95% CIs of means. * Values significantly different
when compared to NGT. (Taken from Fadini et al., 2009)
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
183
4. Clinical application (SE)
Investigation of the clinical potential of ESCM has been limited, largely due to the
significant risk associated with the use of G-CSF, the main stem cell mobilizer used in
clinical trial, for extended periods of time (Bensinger et al., 1996; Shimoda et al., 1993).
Recently, a new stem cell mobilizer (StemEnhance®; SE) has been developed that triggers a
much milder increase in the number of PBSC, but its safety allows for a sustained oral daily
consumption over long periods of time, allowing for safe daily ESCM (Jensen et al., 2007).
In brief, SE is an extract from the cyanophyta Aphanizomenon flos-aquae that concentrates a
protein with an estimated molecular weight of 160–180 kDa, which was shown to be a
selective L-selectin blocker. Oral consumption of 1 gram of SE was been shown to trigger an
average 25% increase in the number of PBSC within 60 minutes after consumption. The
magnitude of the mobilization induced by SE is much smaller than that triggered by G-CSF,
however its safety allows for continuous use and therefore offers a novel approach in the
study of ESCM. To test its therapeutic potential, SE was used in a number of preliminary
clinical trials involving a number of diagnostic entities.
4.1 Parkinson
MC is a 62 year old male with a 17 year history of Parkinson’s Disease. MC was initially
diagnosed with Parkinson’s type resting pill-rolling tremor affecting his left hand and then
progressing to the limbs on left side of his body. Over the course of five years the tremors
increased gradually affecting the right side as well. At ten years into the disease process, MC
was no longer able to practice as an attorney, mobility was affected and limitations
increased compromising his personal and professional abilities.
In 2009, before the treatment of SE, the left side tremors had significantly worsened,
symptoms of stiffness and bradykinesia which introduced a shuffling gait and poor balance
made it unsafe for HM to ambulate without the use of a cane. At the time MC began
consuming SE he was unable to dress himself, put on his watch, write, feed himself or drive
his car. After 45 days on the product, consuming 1 gram of SE, three times per day, MC
showed significant improvement with decreased tremors, less stiffening and bradykinesia;
at this time MC was able to get around without the use of his cane. After 60 days on the
product, judging that the benefits had plateaued, MC discontinued the use of SE. Forty – five
days later, signs of tremor and bradykinesia returned and MC was seeking medical
attention, once again. MC resumed SE with a dose of 2 grams, three times per day and
within six weeks showed much improvement with less tremors, stiffness and bradykinesia.
MC was able to participate in activities of daily living, such as dressing and feeding himself,
he was also able to tie his own tie, put on his watch, and once again, he could ambulate
without using a cane. To date MC has been on SE for two years, he has returned to driving
his car without limitations, he is independent with all activities of daily living and he also
participates in some level of professional activity.
Another patient, MT, is a 52 year old woman with an early onset of Parkinson at age 36,
with tremor as the primary symptom. Early treatment consisted of Pergolid, then Budipin
up to 30 mg three times a day. However due to QTc increase, Budipin was later reduced to
10 mg three times a day. The patient was also treated with Methydopamin 62.5 mg four
times a day. With this treatment, MT’s main problem was the experience of fluctuations and
Tissue Regeneration – From Basic Biology to Clinical Application
184
the beginning of ON-OFF syndrome. One year ago MT began consuming StemEnhance 1
gram three times a day. Today MT’s experiences only minor tremor accompanied with some
dyskinetic syndrome and sometimes propulsion. Her quality of life has increased and she is
much more socially active.
4.2 Traumatic spinal cord injury
A preliminary trial with 8 individuals with spinal cord injuries was performed in a
community center in Hawaii. The cases all involved paraplegia and various degrees of
quadriplegia. Of the 8 cases, 4 dropped due to various circumstances unrelated to the
consumption of SE. Of the 4 remaining participants, 2 had repetitive periods of
hospitalization that made their consumption of SE irregular. Of the two remaining
participants, one experienced mild though significant improvement in mobility while the
other participant experienced significant improvements in mobility.
The latter participant, VS, had a serious car accident 17 years prior to SE consumption and
was left with traumatic brain injury that affected her speech and a significant spinal cord
lesion. At the beginning of the trial, VS was able to lift her right leg approximately 15
centimeter from her chair with no lateral movement, and showed a total absence of
movement in her left leg. She could move her arms, hands and fingers though the
movements were very slow with little dexterity and precision. She had some ability to
move in her bed but was unable to turn herself without assistance. No data was available
to document peripheral sensory perception of the lower limbs or nerve conductivity. After
6 months of daily consumption of 3 grams of SE three times a day, VS could lift her right
leg more than 30 centimeters from her chair, with lateral movement outside of her chair.
She could also lift her left leg off the chair and laterally outside of the chair. VS had less
control over the movement of her left leg, though the magnitude of the movements was
comparable to the movements seen with the right leg. After 10 months, VS was able to
rotate herself in her bed unassisted and from a supine position she could lift both legs to a
90 degree angle and sit in her bed unassisted. Over the period of the trial her upper limbs
also improved in dexterity and her speech showed mild though significant improvement.
VS comes from a disadvantage socio-economic environment and did not have access to
physical therapy beyond the first few years of her injury, she therefore developed leg and
feet deformities that prevented her from bearing weight and possibly resuming physical
therapy.
4.3 Coronary Artery Disease
JP is a 60 year old South African male who experienced a heart attack at 51 years of age.
After diagnosis of Coronary Artery Disease was made a stent placement was performed.
Dietary and lifestyle changes were implemented immediately by JP following the
hospitalization. Unfortunately, 3 years later, JP suffered four additional heart attacks.
During the hospitalization, the angiogram revealed the right and left coronary arteries were
obstructed 100% and 40%, respectively, thus determining that JP was not a good candidate
for bypass surgery. At the time of the last hospitalization, JP had decreased energy and
experienced “stable” angina with any exertion, and his overall quality of life was greatly
compromised. JP was put on a medication regimen which consisted of Atenolol 50mg daily,
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
185
Perindopril 4 mg daily, Elantan 20 mg twice per day, Simvostatin 20 mg daily, Adelat xl 30
mg daily, and Aspirin 1 per day. JP was then put on a list for possible heart transplant.
Three weeks after discharge JP began consuming SE, 1 gram three times per day. After 3
months of taking the product, he received a call for a possible heart transplant and returned
to see his cardiologist for evaluation and comprehensive testing. The cardiologist reported
that JP was making a remarkable recovery, the heart transplant surgery was postponed.
Four months later JP was re-evaluated and found to have made a complete recovery. JP has
since been returning at 6 month intervals for follow up evaluation, and to date he has been
stable with no further coronary incidents. The evaluations and most recent ECG conclude
that JP has normal heart function. Presently, JP shares that he is not on any medications,
blood pressure is 126/65 mm Hg, he continues to take SE and experiences good quality of
life.
4.4 Diabetes and rheumatoid arthritis
NA is a 47 year old Colombian woman who was diagnosed at 18 year of age with deforming
rheumatoid arthritis. Over the years she managed her condition with the use of Prednisone,
Methotrexate, Sulfasalazina, and Diclofenac. Four years ago NA was diagnosed with
diabetes mellitus with glycemia reaching 308 mg/dL and assumed treatment with Euglucon
5 mg twice a day along with NPH insulin. About one year ago, due to her arthritic condition
she became wheelchair bound and required assistance for bathing and dressing. At that time
her medical records show levels of C-Reactive Protein (CRP) of 96.2 mg/L, Erythrocytes
sedimentation rate (ESR) of 81 mm/H, Platelet count of 535 K/ul, fasting glucose of 147
mg/dL and HgbA1c (or Glycosylated Hemoglobin) of 8.07. A few months after becoming
wheelchair bound NA began consuming 1 gram of SE once a day. After one month she
subjectively reported a reduction in pain and inflammation, while 3 months later her
mobility had improved to the point that she could bathe and dress alone. After 6 months she
began using a walker, then switched to a cane and one year later she was walking unaided.
Her last medical records indicate a level of CRP of 2.1 mg/L, ESR of 34 mm/H, Platelet
count of 485 K/ul. NA has also discontinued the use of any diabetes medication; her records
indicate a fasting glucose of 106 mg/dL with a glycemia not exceeding 120 mg/dL and
HgbA1c of 5.62.
At the time of writing this report, NA does not take any anti-inflammatory drug, undergoes
a remarkable improvement in her quality of life, keeping daily consumption for 15
consecutive months of 1 gram of SE, bone support supplementation of 500 mg Calcitriol
(1,25-dihydroxycholecalciferol) every month, milk of magnesia and an annual dose of
Zoledronic Acid.
4.5 Cerebrovascular accident (Stroke)
In September 2008, GE, a 78 year old male surgical oncologist who was otherwise in good
health, had a stroke. The MRI/MRA revealed an acute infarct involving the right lentiform
nucleus, moderately extensive chronic small vessel ischemic changes, chronic lacunar infarct
involving the right ventromedial thalamus, and intracranial atherosclerotic vascular disease.
The stroke left GE with aphasia and a reduced ability to perform any physical activity.
October 2009, 13 months after the stroke, GE began taking SE; GE consumed 1 gram twice
Tissue Regeneration – From Basic Biology to Clinical Application
186
per day and after 8 weeks on the product GE noticed improvement with his speech and
experienced more energy with improved balance. In April 2010 a repeat MRI/MRA showed
no evidence of an acute infarct. GE’s aphasia was completely resolved at this point and his
overall mobility was improved allowing him to perform all activities of daily living. In
January of 2011 a repeat MRI/MRA of the anterior and posterior cerebral arteries
demonstrated no evidence of hemodynamically significant stenosis, and revealed normal
vertebrobasilar arteries with no evidence of intracranial aneurysm or vascular
malformation.
To date, GE has resumed playing tennis at age 81, walks around the mall in the
neighborhood with other senior citizens and has returned to his professional activity as a
surgeon.
4.6 Kidney failure
The whole paragraph should read: “GW is 36 months old; he was born with a
malfunctioning valve in the urethra, compromising the flow of urine at birth. At ten days
old he had surgery to repair the urethra, however, the damage to the kidneys and the
bladder were already evident, caused from the retention of urine at birth. Following the
surgical procedure, scar tissue was observed to the wall of the bladder and one of the
kidneys did not respond, eventually that kidney quit working and began to atrophy. A scar
was also left on his hand caused from an IV being ripped out after surgery. Before SE was
introduced, GW had stopped growing at 15 months of age and multiple health issues were
leading him toward a kidney transplant. Furthermore, GW had been on antibiotics for one
year due to multiple infections. His body was not eliminating fluid as it should, as
evidenced by the worsening of edema resulting in the use of diuretics. GW had purple feet
caused from compromised circulation, dark spots were observed on his back, his eyes were
blood shot and his overall presentation was pale in appearance. GW’s activity was nothing
normal for a 1 ½ year old child; his energy level was very depleted and he was unable to
play like a normal toddler.
GW was 21 months old when SE was introduced at 250 mg per day. After 3 days on SE his
mother reported that his eyes were crystal clear and he was observed running through the
house full of energy. The dark spots on his back began to fade after one week of SE
consumption. Red color was seen on GW cheeks for the first time since he was born. After
75 days on SE, GW had grown 6cm taller and the shrinking kidney at birth had measured
0.8cm larger. GW is off all pharmaceutical medications to date and his bladder and kidney
are back to normal functioning to their fullest capacity, the scar to his hand is completely
gone.
5. Conclusion
The benefits of ESCM on various degenerative conditions have been documented in several
animal models and in humans. In some cases, BMSC clearly migrate into tissues and directly
contribute to the formation of new functional somatic cells of the target tissue. However in
other cases, especially diseases affecting the heart and central nervous system, a primary
mechanism of action appears to be the secretion of paracrines that stimulate the
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
187
proliferation and differentiation of tissue stem cells. Much work remains to be done to
clearly elucidate the mechanisms of action behind the benefits of ECSM in various
diagnostic entities, however from a clinical standpoint it remains that regardless of the
mechanism of action, ESCM appears to be a valuable approach to increase the quality of life
of patients affected by various degenerative diseases.
Various stem cell mobilizers have been documented in the scientific literature and many of
them have been associated with side effects that prevent the application in humans of what
has been documented as effective in various animal models. Such mobilizers include G-CSF,
Stem Cell Factor, interleukin-8 and plerixafor (Lemery et al., 2011), which have all been
associated with side effects going from diarrhea, nausea, pain and numbness to pericarditis
and thrombosis. In spite of the potential benefits, such side effects have largely prevented
the use of such compounds for ESCM in humans, and the lack of safe stem cell mobilizers
largely explains the limited interest so far in this therapeutic approach. The main challenge
in further investigating the therapeutic potential of ESCM remains therefore the
development of safe stem cell mobilizers. In the meanwhile, SE appears to be a valuable tool
to study the clinical benefits of ESCM.
6. References
Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ. (2004) Stromal cell-derived
factor-1alpha plays a critical role in stem cell recruitment to the heart after
myocardial infarction but is not sufficient to induce homing in the absence of
injury. Circulation. 110(21):3300-5.
Abdel-Latif A, Bolli R, Zuba-Surma EK, Tleyjeh IM, Hornung CA, Dawn B. (2008)
Granulocyte colony-stimulating factor therapy for cardiac repair after acute
myocardial infarction: a systematic review and meta-analysis of randomized
controlled trials. Am Heart J. 2008 Aug;156(2):216-226.e9.
Abedi M, Greer DA, Colvin GA. Demers DA, Dooner MS, Harpel JA, Pimentel J, Menon
MK and Quesenberry PJ (2004) Tissue injury in marrow transdifferentiation Blood
Cells Mol Dis 32(1):42 – 46
Arufe MC, De la Fuente A, Fuentes-Boquete I, De Toro FJ, Blanco FJ. (2009) Differentiation
of synovial CD-105(+) human mesenchymal stem cells into chondrocyte-like cells
through spheroid formation. J Cell Biochem. 108(1):145-55.
Arsenijevic Y, Weiss S, Schneider B, Aebischer P. (2001) Insulin-like growth factor-I is
necessary for neural stem cell proliferation and demonstrates distinct actions of
epidermal growth factor and fibroblast growth factor-2. J Neurosci. 21(18):7194-202.
Arvidsson A, T Collin, D Kirik, Z Kokaia and O Lindvall. (2002). Neuronal replacement
from endogenous precursors in the adult brain after stroke. Nature Med 8:963–970.
Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Kiedrowski M, Rovner A, Ellis SG,
Thomas JD, DiCorleto PE, Topol EJ, Penn MS. (2003) Effect of stromal-cell-derived
factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy.
Lancet. 362(9385):697-703.
Avigdor A, Goichberg P, Shivtiel S, Dar A, Peled A, Samira S, Kollet O, Hershkoviz R, Alon
R, Hardan I, Ben-Hur H, Naor D, Nagler A, Lapidot T. (2004) CD44 and hyaluronic
Tissue Regeneration – From Basic Biology to Clinical Application
188
acid cooperate with SDF-1 in the trafficking of human CD34+ stem/progenitor cells
to bone marrow. Blood 103(8):2981-9.
Bachelder RE, Wendt MA, Mercurio AM. (2002) Vascular endothelial growth factor
promotes breast carcinoma invasion in an autocrine manner by regulating the
chemokine receptor CXCR4. Cancer Res 62:7203–7206.
Bagri A, Gurney T, He X, Zou YR, Littman DR, Tessier-Lavigne M, Pleasure SJ. (2002) The
chemokine SDF-1 regulates migration of dentate granule cells. Development
129:4249–4260.
Baumhäkel M, Werner N, Böhm M, Nickenig G. (2006) Circulating endothelial progenitor
cells correlate with erectile function in patients with coronary heart disease. Eur
Heart J. 27(18):2184-8.
Behfar A, Faustino RS, Arrell DK, Dzeja PP, Perez-Terzic C, Terzic A. (2008) Guided stem
cell cardiopoiesis: discovery and translation. J Mol Cell Cardiol. 45(4):523-9. Epub
2008 Sep 26.
Behfar A, Yamada S, Crespo-Diaz R, Nesbitt JJ, Rowe LA, Perez-Terzic C, Gaussin V, Homsy
C, Bartunek J, Terzic A. (2010) Guided cardiopoiesis enhances therapeutic benefit of
bone marrow human mesenchymal stem cells in chronic myocardial infarction. J
Am Coll Cardiol. 56(9):721-34.
Beltrami AP, Urbanek K, Kajstura J, Yan SM, Finato N, Bussani R, Nadal-Ginard B, Silvestri
F, Leri A, Beltrami CA, Anversa P. (2001) Evidence that human cardiac myocytes
divide after myocardial infarction. N Engl J Med 344:1750–1757.
Bensinger W, Buckner W, Rowley S, Storb R, Appelbaum F. (1996) Treatment of normal
donors with recombinant growth factors for transplantation of allogeneic blood
stem cells. Bone Marrow Transplant. 17:S19-21.
Bergmann O, Bhardwaj RD, Bernard S, Zdunek S, Barnabé-Heider F, Walsh S, Zupicich J,
Alkass K, Buchholz BA, Druid H, Jovinge S, and Frisén J. (2009) Evidence for
cardiomyocyte renewal in humans. Science 324: 98-102.
Bonavia R, Bajetto A, Barbero S, Porcile C, Pirani P, Ravetti JL, Zona GL, Spaziante R, Florio
T, Schettini G. (2003) Chemokines and their receptors in the CNS: expression of
CXCL12/SDF-1 and CXCR4 and their role in astrocyte proliferation. Toxicol Lett
139:181–189.
Broxmeyer HE, Kohli L, Kim CH, Lee Y, Mantel C, Cooper S, Hangoc G, Shaheen M, Li X,
Clapp DW. (2003) Stromal cell-derived factor-1/CXCL12 directly enhances
survival/antiapoptosis of myeloid progenitor cell through CXCR4 and G proteins
and enhances engraftment of competitive, repopulating stem cells. J Leukoc Biol
73:630–638.
Broxmeyer HE, Orschell CM, Clapp DW, Hangoc G, Cooper S, Plett PA, Liles WC, Li X,
Graham-Evans B, Campbell TB, Calandra G, Bridger G, Dale DC, Srour EF. (2005)
Rapid mobilization of murine and human hematopoietic stem and progenitor cells
with AMD3100, a CXCR4 antagonist. J Exp Med. 201(8):1307-18.
Burchfield JS, Iwasaki M, Koyanagi M, Urbich C, Rosenthal N, Zeiher AM, Dimmeler S.
(2008) Interleukin-10 from transplanted bone marrow mononuclear cells
contributes to cardiac protection after myocardial infarction. Circ Res 103(2):203-11.
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
189
Can A, Balci D. (2011) Isolation, Culture, and Characterization of Human Umbilical Cord
Stroma-derived Mesenchymal Stem Cells. Methods Mol Biol. 698:51-62.
Carion A, Benboubker L, Hérault O, Roingeard F, Degenne M, Senecal D, Desbois I,
Colombat P, Charbord P, Binet C, Domenech J. (2003) Stromal-derived factor 1 and
matrix metalloproteinase 9 levels in bone marrow and peripheral blood of patients
mobilized by granulocyte colony-stimulating factor and chemotherapy.
Relationship with mobilizing capacity of haematopoietic progenitor cells. Br J
Haematol. 122(6):918-26.
Chandra V, G S, Phadnis S, Nair PD, Bhonde RR. (2009) Generation of pancreatic hormoneexpressing
islet-like cell aggregates from murine adipose tissue-derived stem cells.
Stem Cells. 27(8):1941-53.
Chavey C, Mari B, Monthouel MN, Bonnafous S, Anglard P, Van Obberghen E, Tartare-
Deckert S. (2003) Matrix metalloproteinases are differently expressed in adipose
tissue during obesity and modulate adipocyte differentiation. J Biol Chem
278:11888 –11896.
Chen J, Zhang ZG, Li Y, Wang L, Xu YX, Gautam SC, Lu M, Zhu Z, Chopp M. (2003)
Intravenous administration of human bone marrow stromal cells induces
angiogenesis in the ischemic boundary zone after stroke in rats. Circ Res. 92(6):692-
9.
Chen X, Fang H, Schwob JE. (2004) Multipotency of purified, transplanted globose basal
cells in olfactory epithelium. J Comp Neurol. 469(4):457-74.
Choi JH, Kim KL, Huh W, Kim B, Byun J, Suh W, Sung J, Jeon ES, Oh HY, Kim DK. (2004)
Decreased number and impaired angiogenic function of endothelial progenitor
cells in patients with chronic renal failure. Arterioscler Thromb Vasc Biol.
24(7):1246-52.
Clarris BJ, Fraser JR. (1968) On the pericellular zone of some mammalian cells in vitro. Exp
Cell Res. 49(1):181-93.
Comper WD, Laurent TC. (1978) Physiological function of connective tissue
polysaccharides. Physiol Rev. 58(1):255-315.
Contreras JL, Smyth CA, Eckstein C, Bilbao G, Thompson JA, Young CJ, Eckhoff DE. (2003)
Peripheral mobilization of recipient bone marrow-derived endothelial progenitor
cells enhances pancreatic islet revascularization and engraftment after intraportal
transplantation. Surgery 134(2):390-8.
Cottler-Fox MH, Lapidot T, Petit I, Kollet O, DiPersio JF, Link D, Devine S. (2003) Stem cell
mobilization. Hematology Am Soc Hematol Educ Program. 419-37.
Croop JM, Cooper R, Fernandez C, Graves V, Kreissman S, Hanenberg H, Smith FO,
Williams DA. (2001) Mobilization and collection of peripheral blood CD34+ cells
from patients with Fanconi anemia. Blood. 98(10):2917-21.
Dasgupta C, Escobar-Poni B, Shah M, Duncan J, Fletcher WH. (1999) Misregulation of
connexin43 gap junction channels and congenital heart defects. Novartis Found
Symp. 219:212-21.
Deasy BM, Qu-Peterson Z, Greenberger JS, Huard J. (2002) Mechanisms of muscle stem cell
expansion with cytokines. Stem Cells. 20(1):50-60.
Tissue Regeneration – From Basic Biology to Clinical Application
190
De Clercq E. (2005) Potential clinical applications of the CXCR4 antagonist bicyclam
AMD3100. Mini Rev Med Chem. 5(9):805-24.
Diller GP, van Eijl S, Okonko DO, Howard LS, Ali O, Thum T, Wort SJ, Bédard E, Gibbs JS,
Bauersachs J, Hobbs AJ, Wilkins MR, Gatzoulis MA, Wharton J. (2008) Circulating
endothelial progenitor cells in patients with Eisenmenger syndrome and idiopathic
pulmonary arterial hypertension. Circulation 117(23):3020-30.
Docherty K. (2009) Pancreatic stellate cells can form new beta-like cells. Biochem J. 421(2):
e1-4.
Dos Santos F, Andrade PZ, Eibes G, da Silva CL, Cabral JM. (2011) Ex Vivo Expansion of
Human Mesenchymal Stem Cells on Microcarriers. Methods Mol Biol. 698:189-198.
Drapeau C, Antarr D, Ma H, Yang Z, Tang L, Hoffman RM, Schaeffer DJ. (2010)
Mobilization of bone marrow stem cells with StemEnhance improves muscle
regeneration in cardiotoxin-induced muscle injury. Cell Cycle 9(9):1819-23.
Dunac A, Frelin C, Popolo-Blondeau M, Chatel M, Mahagne MH, Philip PJ. (2007)
Neurological and functional recovery in human stroke are associated with
peripheral blood CD34+ cell mobilization. J Neurol. 2007 Mar;254(3):327-32.
Eizawa T, Murakami Y, Matsui K, Takahashi M, Muroi K, Amemiya M, Takano R, Kusano
E, Shimada K, Ikeda U. (2003) Circulating endothelial progenitor cells are reduced
in hemodialysis patients. Curr Med Res Opin. 19(7):627-33.
Ellis SG, Penn MS, Bolwell B, Garcia M, Chacko M, Wang T, Brezina KJ, McConnell G, Topol
EJ. (2006) Granulocyte colony stimulating factor in patients with large acute
myocardial infarction: results of a pilot dose-escalation randomized trial. Am Heart
J. 152(6):1051.e9-14.
Fadini GP, de Kreutzenberg S, Agostini C, Boscaro E, Tiengo A, Dimmeler S, Avogaro A
(2009) Low CD34+ cell count and metabolic syndrome synergistically increase the risk of
adverse outcomes. Atherosclerosis 2009; 207: 213– 219
Fadini GP, Boscaro E, de Kreutzenberg S, Agostini C, Seeger F, Dimmeler S, Zeiher A,
Tiengo A, Avogaro A. (2010) Time course and mechanisms of circulating
progenitor cell reduction in the natural history of type 2 diabetes. Diabetes Care.
2010 May;33(5):1097-102.
Fallon J, S Reid, R Kinyamu, I Opole, R Opole, J Baratta, M Korc, TL Endo, A Duong, G
Nguyen, M Karkehabadhi, D Twardzik, S Patel and S Loughlin. (2000). In vivo
induction of massive proliferation, directed migration, and differentiation of neural
cells in the adult mammalian brain. PNAS 96:14686–14691.
Ferrari G, Cusella-De Angelis G, Coletta M, Paolucci E, Stornaiuolo A, Cossu G, Mavilio F.
(1998) Muscle regeneration by bone marrow-derived myogenic progenitors.
Science. 279(5356):1528-30.
Fibbe WE, Pruijt JF, Velders GA, et al. (1999) Biology of IL-8-induced stem cell mobilization.
Ann N Y Acad Sci. 872:71-82.
Fox A, Smythe J, Fisher N, Tyler MP, McGrouther DA, Watt SM, Harris AL. (2008)
Mobilization of endothelial progenitor cells into the circulation in burned patients.
Br J Surg. 95(2):244-51
Fraser JR, Laurent TC, Laurent UB. (1997) Hyaluronan: its nature, distribution, functions
and turnover. J Intern Med. 242(1):27-33.
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
191
Friedl P, Maaser K, Klein CE, Niggemann B, Krohne G, Zanker KS. Migration of highly
aggressive MV3 melanoma cells in 3-dimensional collagen lattices results in local
matrix reorganization and shedding of alpha2 and beta1 integrins and CD44.
Cancer Res.57:2061-2070.
Fukuda S, Bian H, King AG, Pelus LM. (2007) The chemokine GRObeta mobilizes early
hematopoietic stem cells characterized by enhanced homing and engraftment. Blood
110(3):860-9.
Fukuhara S, Tomita S, Nakatani T, Ohtsu Y, Ishida M, Yutani C, Kitamura S. (2004) G-CSF
promotes bone marrow cells to migrate into infarcted mice heart, and differentiate
into cardiomyocytes. Cell Transplant. 13(7-8):741-8
Gao X, Song L, Shen K, Wang H, Niu W, Qin X. (2008) Transplantation of bone marrow
derived cells promotes pancreatic islet repair in diabetic mice. Biochem Biophys
Res Commun. 371(1):132-7.
Gnecchi M, He H, Liang OD, Melo LG, Morello F, Mu H, Noiseux N, Zhang L, Pratt RE,
Ingwall JS, Dzau VJ. (2005) Paracrine action accounts for marked protection of
ischemic heart by Akt-modified mesenchymal stem cells. Nat Med. 11(4):367-8.
Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, Mu H, Melo LG, Pratt RE,
Ingwall JS, Dzau VJ. (2006) Evidence supporting paracrine hypothesis for Aktmodified
mesenchymal stem cell-mediated cardiac protection and functional
improvement. FASEB J. 20(6):661-9.
Gordon MK, Sher D, Karrison T, Kebriaei P, Chuang K, Zhang Y, McDonnell D, Artz A,
Godley L, Odenike O, Rich E, Michaelis L, Thirman MJ, Wickrema A, van Besien K,
Larson RA, Stock W. (2008) Successful autologous stem cell collection in patients
with chronic myeloid leukemia in complete cytogenetic response, with quantitative
measurement of BCR-ABL expression in blood, marrow, and apheresis products.
Leuk Lymphoma. 49(3):531-7.
Greco SJ, Zhou C, Ye JH, Rameshwar P. (2008) A method to generate human mesenchymal
stem cell-derived neurons which express and are excited by multiple
neurotransmitters. Biol Proced Online. 10:90-101.
Grisar J, Aletaha D, Steiner CW, Kapral T, Steiner S, Seidinger D, Weigel G, Schwarzinger I,
Wolozcszuk W, Steiner G, Smolen JS. (2005) Depletion of endothelial progenitor
cells in the peripheral blood of patients with rheumatoid arthritis. Circulation.
111(2):204-11.
Gronthos S, Arthur A, Bartold PM, Shi S. (2011) A method to isolate and culture expand
human dental pulp stem cells. Methods Mol Biol. 698:107-21.
Hahn JY, Cho HJ, Kang HJ, Kim TS, Kim MH, Chung JH, Bae JW, Oh BH, Park YB, Kim HS.
(2008) Pre-treatment of mesenchymal stem cells with a combination of growth
factors enhances gap junction formation, J Am Coll Cardiol. 51(9):933-43.
Hasegawa Y, Ogihara T, Yamada T, Ishigaki Y, Imai J, Uno K, Gao J, Kaneko K, Ishihara H,
Sasano H, Nakauchi H, Oka Y, Katagiri H. (2007) Bone marrow (BM)
transplantation promotes beta-cell regeneration after acute injury through BM cell
mobilization. Endocrinology. 148(5):2006-15.
Tissue Regeneration – From Basic Biology to Clinical Application
192
Hatch HM, Zheng D, Jorgensen ML, Petersen BE. (2002) SDF-1alpha/CXCR4: a mechanism
for hepatic oval cell activation and bone marrow stem cell recruitment to the
injured liver of rats. Cloning Stem Cells. 4(4):339-51.
Hayakawa J, Migita M, Ueda T, Shimada T, Fukunaga Y. (2003) Generation of a chimeric
mouse reconstituted with green fluorescent protein-positive bone marrow cells: a
useful model for studying the behavior of bone marrow cells in regeneration in
vivo. Int J Hematol. 77(5):456-62.
Heissig B, Hattori K, Dias S, Friedrich M, Ferris B, Hackett NR, Crystal RG, Besmer P, Lyden
D, Moore MA, Werb Z, Rafii S. (2002) Recruitment of stem and progenitor cells
from the bone marrow niche requires MMP-9 mediated release of kit-ligand. Cell.
109:625-637.
Hennemann B, Ickenstein G, Sauerbruch S, Luecke K, Haas S, Horn M, Andreesen R,
Bogdahn U, Winkler J. (2008) Mobilization of CD34+ hematopoietic cells, colonyforming
cells and long-term culture-initiating cells into the peripheral blood of
patients with an acute cerebral ischemic insult. Cytotherapy. 10(3):303-11.
Henschler R, Deak E, Seifried E.(2008) Homing of Mesenchymal Stem Cells. Transfus Med
Hemother. 35(4):306-312.
Herbrig K, Haensel S, Oelschlaegel U, Pistrosch F, Foerster S, Passauer J. (2006) Endothelial
dysfunction in patients with rheumatoid arthritis is associated with a reduced
number and impaired function of endothelial progenitor cells. Ann Rheum Dis.
65(2):157-63.
Herbrig K, Pistrosch F, Oelschlaegel U, Wichmann G, Wagner A, Foerster S, Richter S, Gross
P, Passauer J. (2004) Increased total number but impaired migratory activity and
adhesion of endothelial progenitor cells in patients on long-term hemodialysis. Am
J Kidney Dis. 44(5):840-9.
Hess DC, Hill WD, Martin-Studdard A, Carroll J, Brailer J, Carothers J. (2002) Bone marrow
as a source of endothelial cells and NeuN-expressing cells after stroke. Stroke
33(5):1362-8.
Hisashi Y, Tomita S, Nakatani T, Fukuhara S, Yutani C, Kitamura S. (2004) Granulocytecolony
stimulating factor enhanced the recruitment of bone marrow cells into the
heart: time course evaluation of phenotypic differentiation in the doxorubicininduced
cardiomyopathic model. Jpn J Thorac Cardiovasc Surg. 52(10):451-5.
Hoehn M, Küstermann E, Blunk J, Wiedermann D, Trapp T, Wecker S, Föcking M, Arnold
H, Hescheler J, Fleischmann BK, Schwindt W, Bührle C. (2002) Monitoring of
implanted stem cell migration in vivo: a highly resolved in vivo magnetic
resonance imaging investigation of experimental stroke in rat. Proc Natl Acad Sci U
S A. 99(25):16267-72.
Hofmann NA, Reinisch A, Strunk D. (2009) Isolation and large scale expansion of adult
human endothelial colony forming progenitor cells. J Vis Exp. 28;(32).
Hwang JH, Kim SW, Park SE, Yun HJ, Lee Y, Kim S, Jo DY. (2006) Overexpression of
stromal cell-derived factor-1 enhances endothelium-supported transmigration,
maintenance, and proliferation of hematopoietic progenitor cells. Stem Cells Dev.
15(2):260-8.
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
193
Ince H, Petzsch M, Kleine HD, Eckard H, Rehders T, Burska D, Kische S, Freund M,
Nienaber CA. (2005) Prevention of left ventricular remodeling with granulocyte
colony-stimulating factor after acute myocardial infarction: final 1-year results of
the Front-Integrated Revascularization and Stem Cell Liberation in Evolving Acute
Myocardial Infarction by Granulocyte Colony-Stimulating Factor (FIRSTLINEAMI)
Trial. Circulation 112(9 Suppl):I73-80.
Insausti CL, Blanquer M, Meseguer L, López-Martínez MC, Ferez X, Rodriguez FJ, Cabañas
V, Funes C, Nicolás FJ, Majado MJ, Moraleda JM. Isolation and characterization of
mesenchymal stem cells from the fat layer on the density gradient separated bone
marrow. Stem Cells Dev. 2011 Apr 19. [Epub ahead of print]
Ivanovic Z, Duchez P, Chevaleyre J, Vlaski M, Lafarge X, Dazey B, Robert-Richard E,
Mazurier F, Boiron JM. (2011) Clinical-scale cultures of cord blood CD34+ cells to
amplify committed progenitors and maintain stem cell activity. Cell Transplant.
2011 Feb 3. [Epub ahead of print]
Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, Silver M, Li T, Isner
JM, Asahara T. (2002) Endothelial progenitor cell vascular endothelial growth
factor gene transfer for vascular regeneration. Circulation 105:732–738.
Jackson KA, Majka SM, Wang H, Pocius J, Hartley CJ, Majesky MW, Entman ML, Michael
LH, Hirschi KK, Goodell MA. (2001) Regeneration of ischemic cardiac muscle and
vascular endothelium by adult stem cells. J Clin Invest. 107(11):1395-402.
Jang YY, Collector MI, Baylin SB, Diehl AM, Sharkis SJ. (2004) Hematopoietic stem cells
convert into liver cells within days without fusion. Nat Cell Biol. 6(6):532-9.
Janowska-Wieczorek A, Marquez LA, Nabholtz JM, Cabuhat ML, Montaño J, Chang H,
Rozmus J, Russell JA, Edwards DR, Turner AR. (1999) Growth factors and
cytokines upregulate gelatinase expression in bone marrow CD34(+) cells and their
transmigration through reconstituted basement membrane. Blood. 93:3379-3390.
Janowska-Wieczorek A, Marquez LA, Dobrowsky A, Ratajczak MZ, Cabuhat ML. (2000)
Differential MMP and TIMP production by human marrow and peripheral blood
CD34- cells in response to chemokines. Exp Hematol 28:1274–1285.
Jensen GS, Hart AN, Zaske LA, Drapeau C, Gupta N, Schaeffer DJ, Cruickshank JA. (2007)
Mobilization of human CD34+ CD133+ and CD34+ CD133(-) stem cells in vivo by
consumption of an extract from Aphanizomenon flos-aquae–related to modulation
of CXCR4 expression by an L-selectin ligand? Cardiovasc Revasc Med. 8(3):189-202.
Jin FY, Qiu LG, Li QC, Meng HX, Wang YF, Yu Z, Li Q, Han JL. (2006) The effect of matrix
metalloproteinase-9 in granulocyte colony stimulation factor-induced stem cell
mobilization. Zhonghua Yi Xue Za Zhi. 86(42):2966-70.
Jo DY, Rafii S, Hamada T, Moore MA. (2000) Chemotaxis of primitive hematopoietic cells in
response to stromal cell-derived factor-1. J Clin Invest. 105(1):101-11.
Junhui Z, Xingxiang W, Guosheng F, Yunpeng S, Furong Z, Junzhu C. (2008) Reduced
number and activity of circulating endothelial progenitor cells in patients with
idiopathic pulmonary arterial hypertension. Respir Med. 102(7):1073-9.
Kajstura J, Leri A, Finato N, Di Loreto C, Beltrami CA, Anversa P. (1998) Myocyte
proliferation in end-stage cardiac failure in humans. Proc Natl Acad Sci USA
95:8801–8805.
Tissue Regeneration – From Basic Biology to Clinical Application
194
Kamihata H, Matsubara H, Nishiue T, Fujiyama S, Tsutsumi Y, Ozono R, Masaki H, Mori Y,
Iba O, Tateishi E, Kosaki A, Shintani S, Murohara T, Imaizumi T, Iwasaka T. (2001)
Implantation of bone marrow mononuclear cells into ischemic myocardium
enhances collateral perfusion and regional function via side supply of angioblasts,
angiogenic ligands, and cytokines. Circulation. 104(9):1046-52.
Kan I, Melamed E, Offen D. (2005) Integral therapeutic potential of bone marrow
mesenchymal stem cells. Curr Drug Targets. 6(1):31-41.
Kang S, Yang Y, Li CJ, Gao R. (2007) Effectiveness and tolerability of administration of
granulocyte colony-stimulating factor on left ventricular function in patients with
myocardial infarction: a meta-analysis of randomized controlled trials. Clin Ther.
2007 Nov;29(11):2406-18.
Karra R, Wu SM. (2008) Multipotent stem cells in cardiac regenerative therapy. Regen Med.
3(2):189-98.
Kawada H, Takizawa S, Takanashi T, Morita Y, Fujita J, Fukuda K, Takagi S, Okano H,
Ando K, Hotta T. (2006) Administration of hematopoietic cytokines in the subacute
phase after cerebral infarction is effective for functional recovery facilitating
proliferation of intrinsic neural stem/progenitor cells and transition of bone
marrow-derived neuronal cells. Circulation. 113(5):701-10.
Keilhoff G, Stang F, Goihl A, Wolf G, Fansa H. (2006) Transdifferentiated mesenchymal stem
cells as alternative therapy in supporting nerve regeneration and myelination. Cell
Mol Neurobiol. 26(7-8):1235-52.
King AG, Horowitz D, Dillon SB, Levin R, Farese AM, MacVittie TJ, Pelus LM. (2001) Rapid
mobilization of murine hematopoietic stem cells with enhanced engraftment
properties and evaluation of hematopoietic progenitor cell mobilization in rhesus
monkeys by a single injection of SB-251353, a specific truncated form of the human
CXC chemokine GRObeta. Blood. 97(6):1534-42.
Kinnaird T, Stabile E, Burnett MS, Shou M, Lee CW, Barr S, Fuchs S, Epstein SE. (2004) Local
delivery of marrow-derived stromal cells augments collateral perfusion through
paracrine mechanisms. Circulation. 109(12):1543-9.
Kleeberger W, Versmold A, Rothämel T, Glöckner S, Bredt M, Haverich A, Lehmann U,
Kreipe H. (2003) Increased chimerism of bronchial and alveolar epithelium in
human lung allografts undergoing chronic injury. Am J Pathol. 162(5):1487-94.
Klein JD, Fauza DO. (2011) Amniotic and placental mesenchymal stem cell isolation and
culture. Methods Mol Biol. 698:75-88.
Kolbe M, Dohle E, Katerla D, Kirkpatrick CJ, Fuchs S. (2010) Enrichment of outgrowth
endothelial cells in high and low colony-forming cultures from peripheral blood
progenitors. Tissue Eng Part C Methods. 16(5):877-86.
Kollet O, Shivtiel S, Chen YQ, Suriawinata J, Thung SN, Dabeva MD, Kahn J, Spiegel A, Dar
A, Samira S, Goichberg P, Kalinkovich A, Arenzana-Seisdedos F, Nagler A, Hardan
I, Revel M, Shafritz DA, Lapidot T. (2003) HGF, SDF-1, and MMP-9 are involved in
stress-induced human CD34+ stem cell recruitment to the liver. J Clin Invest.
112(2):160-9.
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
195
Krause DS, Theise ND, Collector MI, Henegariu O, Hwang S, Gardner R, Neutzel S, and
Sharkis SJ (2001) Multi-organ, multi-lineage engraftment by a single bone marrowderived
stem cell. Cell 105:369-377
Kucia M, Ratajczak J, Reca R, Janowska-Wieczorek A, Ratajczak MZ.. (2004) Tissue-specific
muscle, neural and liver stem/progenitor cells reside in the bone marrow, respond
to an SDF-1 gradient and are mobilized into peripheral blood during stress and
tissue injury. Blood Cells Mol Dis. 32(1):52–57.
Laflamme MA, Myerson D, Saffitz JE, Murry CE. (2002) Evidence for cardiomyocyte
repopulation by extracardiac progenitors in transplanted human hearts. Circ Res.
90(6):634-40.
Lapid K, Vagima Y, Kollet O, Lapidot T. (2009) Egress and mobilization of hematopoietic
stem and progenitor cells. StemBook. Cambridge (MA): Harvard Stem Cell
Institute; 2008-.2009 Mar 15.
Lapidot T and Petit I. (2002) Current understanding of stem cell mobilization: the roles of
chemokines, proteolytic enzymes, adhesion molecules, cytokines, and stromal cells.
Exp Hematol. 30(9):973-81.
Lazarini F, Tham TN, Casanova P, Arenzana-Seisdedos F, Dubois-Dalcq M. (2003) Role of
the alpha-chemokine stromal cell-derived factor (SDF-1) in the developing and
mature central nervous system. Glia 2003;42:139–148.
Lee DY, Cho TJ, Kim JA, Lee HR, Yoo WJ, Chung CY, Choi IH. (2008) Mobilization of
endothelial progenitor cells in fracture healing and distraction osteogenesis Bone.
42(5):932-41.
Lee HJ, Kim KS, Park IH, Kim SU (2007) Human neural stem cells over-expressing VEGF
provide neuroprotection, angiogenesis and functional recovery in mouse stroke
model. PLoS ONE 2(1): e156.
Lee KD, Kuo TK, Whang-Peng J, Chung YF, Lin CT, Chou SH, Chen JR, Chen YP and Lee
OK. (2004) In vitro hepatic differentiation of human mesenchymal stem cells.
Hepatology 40:1275-1284.
Lee ST, Chu K, Jung KH, Ko SY, Kim EH, Sinn DI, Lee YS, Lo EH, Kim M, Roh JK. (2005)
Granulocyte colony-stimulating factor enhances angiogenesis after focal cerebral
ischemia. Brain Res. 1058(1-2):120-128.
Lee ST, Chu K, Jung KH, Kim DH, Kim EH, Choe VN, Kim JH, Im WS, Kang L, Park JE,
Park HJ, Park HK, Song EC, Lee SK, Kim M, Roh JK. (2008) Decreased number and
function of endothelial progenitor cells in patients with migraine. Neurology.
70(17):1510-7.
Lemery SJ, Hsieh MM, Smith A, Rao S, Khuu HM, Theresa D, Viano JM, Cook L, Goodwin
R, Boss C, Calandra G, Geller N, Tisdale J, Childs R. (2011) A pilot study evaluating
the safety and CD34+ cell mobilizing activity of escalating doses of plerixafor in
healthy volunteers. Br J Haematol. 153(1):66-75.
Leone AM, Rutella S, Bonanno G, Contemi AM, de Ritis DG, Giannico MB, Rebuzzi AG,
Leone G, Crea F. (2006) Endogenous G-CSF and CD34+ cell mobilization after acute
myocardial infarction. Int J Cardiol. 111(2):202-8.
Leone AM, Galiuto L, Garramone B, Rutella S, Giannico MB, Brugaletta S, Perfetti M, Liuzzo
G, Porto I, Burzotta F, Niccoli G, Biasucci LM, Leone G, Rebuzzi AG, Crea F. (2007)
Tissue Regeneration – From Basic Biology to Clinical Application
196
Usefulness of granulocyte colony-stimulating factor in patients with a large
anterior wall acute myocardial infarction to prevent left ventricular remodeling
(the rigenera study). Am J Cardiol. 100(3):397-403.
Li Y, Chen J, Chen XG, Wang L, Gautam SC, Xu YX, Katakowski M, Zhang LJ, Lu M,
Janakiraman N, Chopp M. (2002) Human marrow stromal cell therapy for stroke in
rat: neurotrophins and functional recovery. Neurology. 59(4):514-23.
Lima C, Escada P, Pratas-Vital J, Branco C, Arcangeli CA, Lazzeri G, Maia CA, Capucho C,
Hasse-Ferreira A, Peduzzi JD. (2010) Olfactory mucosal autografts and
rehabilitation for chronic traumatic spinal cord injury. Neurorehabil Neural Repair.
24(1):10-22.
Loewenstein WR, Rose B. (1992) The cell-cell channel in the control of growth. Semin Cell
Biol. 3(1):59-79.
Mannello F, Tonti GA, Bagnara GP, Papa S. (2006) Role and function of matrix
metalloproteinases in the differentiation and biological characterization of
mesenchymal stem cells. Stem Cells. 24(3):475-81.
Mansilla E, Marín GH, Drago H, Sturla F, Salas E, Gardiner C, Bossi S, Lamonega R,
Guzmán A, Nuñez A, Gil MA, Piccinelli G, Ibar R, Soratti C. (2006) Bloodstream
cells phenotypically identical to human mesenchymal bone marrow stem cells
circulate in large amounts under the influence of acute large skin damage: new
evidence for their use in regenerative medicine. Transplant Proc. 38(3):967-9.
Marchesi C, Belicchi M, Meregalli M, Farini A, Cattaneo A, Parolini D, Gavina M, Porretti L,
D’Angelo MG, Bresolin N, Cossu G, Torrente Y. (2008) Correlation of circulating
CD133+ progenitor subclasses with a mild phenotype in Duchenne muscular
dystrophy patients. PLoS ONE. 3(5):e2218.
Magavi SS and JD Macklis. (2002). Immunocytochemical analysis of neuronal
differentiation. Meth Mol Biol 198:291–297.
Mathews V, Hanson PT, Ford E, Fujita J, Polonsky KS, Graubert TA. (2004) Recruitment of
bone marrow-derived endothelial cells to sites of pancreatic beta-cell injury.
Diabetes 53(1):91-8.
Matsumoto T, Mifune Y, Kawamoto A, Kuroda R, Shoji T, Iwasaki H, Suzuki T, Oyamada A,
Horii M, Yokoyama A, Nishimura H, Lee SY, Miwa M, Doita M, Kurosaka M,
Asahara T. (2008) Fracture induced mobilization and incorporation of bone
marrow-derived endothelial progenitor cells for bone healing. J Cell Physiol.
215(1):234-42
Mezey E, Key S, Vogelsang G, Szalayova I, Lange GD and Crain G. (2003). Transplanted
bone marrow generates new neurons in human brains. Proc. Natl. Acad. Sci. USA
100, 1364-1369.
Middleton J, Neil S, Wintle J, Clark-Lewis I, Moore H, Lam C, Auer M, Hub E, Rot A. (1997)
Transcytosis and surface presentation of IL-8 by venular endothelial cells. Cell.
91(3):385-95.
Mirotsou M, Jayawardena TM, Schmeckpeper J, Gnecchi M, Dzau VJ. (2011) Paracrine
mechanisms of stem cell reparative and regenerative actions in the heart. J Mol Cell
Cardiol. 50(2):280-9.
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
197
Mishra SK, Braun N, Shukla V, Füllgrabe M, Schomerus C, Korf HW, Gachet C, Ikehara Y,
Sévigny J, Robson SC, Zimmermann H. (2006) Extracellular nucleotide signaling in
adult neural stem cells: synergism with growth factor-mediated cellular
proliferation. Development. 133(4):675-84.
Moonen JR, de Leeuw K, van Seijen XJ, Kallenberg CG, van Luyn MJ, Bijl M, Harmsen MC.
(2007) Reduced number and impaired function of circulating progenitor cells in
patients with systemic lupus erythematosus. Arthritis Res Ther. 2007;9(4):R84.
Müller P, Pfeiffer P, Koglin J, Schäfers HJ, Seeland U, Janzen I, Urbschat S, Böhm M. (2002)
Cardiomyocytes of noncardiac origin in myocardial biopsies of human
transplanted hearts. Circulation. 106(1):31-5.
Nakatomi H, T Kuriu, S Okabe, S Yamamoto, O Hatano, N Kawahara, A Tamura, T Kirino
and M Nakafuku. (2002) Regeneration of hippocampal pyramidal neurons after
ischemic brain injury by recruitment of endogenous neural progenitors. Cell
110:429-441.
Neijssen J, Herberts C, Drijfhout JW, Reits E, Janssen L, Neefjes J. (2005) Cross-presentation
by intercellular peptide transfer through gap junctions. Nature. 434(7029):83-8.
Neuss S, Becher E, Wöltje M, Tietze L, Jahnen-Dechent W. (2004) Functional expression of
HGF and HGF receptor/c-met in adult human mesenchymal stem cells suggests a
role in cell mobilization, tissue repair, and wound healing. Stem Cells 22:405– 414.
Nygren JM, Jovinge S, Breitbach M, Säwén P, Röll W, Hescheler J, Taneera J, Fleischmann
BK, Jacobsen SE. (2004) Bone marrow-derived hematopoietic cells generate
cardiomyocytes at a low frequency through cell fusion, but not transdifferentiation.
Nat Med.10(5):494-501.
Okamoto I, Kawano Y, Tsuiki H, Sasaki J, Nakao M, Matsumoto M, Suga M, Ando M,
Nakajima M, Saya H. (1999) CD44 cleavage induced by a membrane-associated
metalloprotease plays a critical role in tumor cell migration. Oncogene. 18:1435-
1446.
Orlic, D., Kajstura, J., Chimenti, S., Jakoniuk, I., Pickel, J., McKay, R., Nadal-Ginard, B.,
Bodine, D. M., Leri, A. & Anversa, P. (2001a) Bone marrow cells regenerate
infracted myocardium. Nature (London) 410, 701–705.
Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, Nadal-Ginard B, Bodine DM,
Leri A, Anversa P. (2001b) Mobilized bone marrow cells repair the infarcted heart,
improving function and survival. Proc Natl Acad Sci U S A. 98(18):10344-9.
Osada T, Watanabe M, Hasuo A, Imai M, Suyama K, Sakai D, Kawada H, Matsumae M,
Mochida J. (2010) Efficacy of the coadministration of granulocyte colonystimulating
factor and stem cell factor in the activation of intrinsic cells after spinal
cord injury in mice. J Neurosurg Spine. 13(4):516-23.
Paczkowska E, Larysz B, Rzeuski R, Karbicka A, Jałowiński R, Kornacewicz-Jach Z,
Ratajczak MZ, Machaliński B. (2005) Human hematopoietic stem/progenitorenriched
CD34(+) cells are mobilized into peripheral blood during stress related to
ischemic stroke or acute myocardial infarction. Eur J Haematol. 75(6):461-7.
Pavlichenko N, Sokolova I, Vijde S, Shvedova E, Alexandrov G, Krouglyakov P, Fedotova O,
Gilerovich EG, Polyntsev DG, Otellin VA Mesenchymal stem cells transplantation
Tissue Regeneration – From Basic Biology to Clinical Application
198
could be beneficial for treatment of experimental ischemic stroke in rats. Brain Res.
2008 Jul 14.
Peled A, Grabovsky V, Habler L, Sandbank J, Arenzana-Seisdedos F, Petit I, Ben-Hur H,
Lapidot T, Alon R. (1999) The chemokine SDF-1 stimulates integrin-mediated arrest
of CD34(+) cells on vascular endothelium under shear flow. J Clin Invest.
104(9):1199-211.
Peled A, Kollet O, Ponomaryov T, Petit I, Franitza S, Grabovsky V, Slav MM, Nagler A,
Lider O, Alon R, Zipori D, Lapidot T. (2000) The chemokine SDF-1 activates the
integrins LFA-1, VLA-4, and VLA-5 on immature human CD34(+) cells: role in
transendothelial/stromal migration and engraftment of NOD/SCID mice. Blood.
95(11):3289-96.
Pereira RF, O’Hara MD, Laptev AV, Halford KW, Pollard MD, Class R, Simon D, Livezey K
and Prockop DJ. (1998) Marrow stromal cells as a source of progenitor cells for
nonhematopoietic tissues in transgenic mice with a phenotype of osteogenesis
imperfecta. Proc. Natl. Acad. Sci. USA 95, 1142-1147.
Peterson DA. (2002). Stem cells in brain plasticity and repair. Curr Opin Pharmacol 2:34–42.
Petit I, Szyper-Kravitz M, Nagler A, Lahav M, Peled A, Habler L, Ponomaryov T, Taichman
RS, Arenzana-Seisdedos F, Fujii N, Sandbank J, Zipori D, Lapidot T. (2003) G-CSF
induces stem cell mobilization by decreasing bone marrow SDF-1 and upregulating
CXCR4. Nat Immunol. 3(7):687-94.
Pineault N, Cortin V, Boyer L, Garnier A, Robert A, Thérien C, Roy DC. (2011) Individual
and synergistic cytokine effects controlling the expansion of cord blood CD34(+)
cells and megakaryocyte progenitors in culture. Cytotherapy. 13(4):467-80.
Quaini F, Urbanek K, Beltrami AP, Finato N, Beltrami CA, Nadal-Ginard B, Kajstura J, Leri
A, Anversa P. (2002) Chimerism of the transplanted heart. N Engl J Med. 346(1):5-
15.
Ratajczak MZ, Majka M, Kucia M, Drukala J, Pietrzkowski Z, Peiper S, Janowska-Wieczorek
A. (2003) Expression of functional CXCR4 by muscle satellite cells and secretion of
SDF-1 by muscle-derived fibroblasts is associated with the presence of both muscle
progenitors in bone marrow and hematopoietic stem/progenitor cells in muscles.
Stem Cells. 21(3):363-71.
Reed RK, Lilja K, Laurent TC. (1988) Hyaluronan in the rat with special reference to the skin.
Acta Physiol Scand. 134(3):405-11.
Ries C, Egea V, Karow M, Kolb H, Jochum M, Neth P. (2007) MMP-2, MT1-MMP, and TIMP-
2 are essential for the invasive capacity of human mesenchymal stem cells:
differential regulation by inflammatory cytokines. Blood. 109(9):4055-63.
Ripa RS, Kastrup J. (2008) G-CSF therapy with mobilization of bone marrow stem cells for
myocardial recovery after acute myocardial infarction–a relevant treatment? Exp
Hematol. 36(6):681-6.
Rustom A, Saffrich R, Markovic I, Walther P, Gerdes HH. (2004) Nanotubular highways for
intercellular organelle transport. Science. 303(5660):1007-10.
Sanchez-Ramos JR. (2002) Neural cells derived from adult bone marrow and umbilical cord
blood. J Neurosci Res. 69(6):880-93
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
199
Schioppa T, Uranchimeg B, Saccani A, Biswas SK, Doni A, Rapisarda A, Bernasconi S,
Saccani S, Nebuloni M, Vago L, Mantovani A, Melillo G, Sica A. (2003) Regulation
of the chemokine receptor CXCR4 by hypoxia. J Exp Med 2003;198:1391–1402.
Schmidt W and KG Reymann. (2002). Proliferating cells differentiate into neurons in the
hippocampal CAI region of gerbils after global cerebral ischemia. Neurosci Lett
334:153–156.
Schrader AJ, Lechner O, Templin M, Dittmar KE, Machtens S, Mengel M, Probst-Kepper M,
Franzke A, Wollensak T, Gatzlaff P, Atzpodien J, Buer J, Lauber J. (2002)
CXCR4/CXCL12 expression and signalling in kidney cancer. Br J Cancer.
86(8):1250-6.
Schwartz Y and Kornowski R. (2003) Autologous stem cells for functional myocardial repair.
Heart Fail Rev. 8(3):237-45.
Sesti C, Hale SL, Lutzko C, Kloner RA. (2005) Granulocyte colony-stimulating factor and
stem cell factor improve contractile reserve of the infarcted left ventricle
independent of restoring muscle mass. J Am Coll Cardiol. 46(9):1662-9.
Shimoda K, Okamura S, Haarada N, Kondo S, Niho Y. (1993) Identification of a functional
receptor for granulocyte colony-stimulating factor on platelets. J Clin Invest.
91:1310-1313.
Shintani S, Murohara T, Ikeda H, Ueno T, Honma T, Katoh A, Sasaki K, Shimada T, Oike Y,
Imaizumi T. (2001) Mobilization of endothelial progenitor cells in patients with
acute myocardial infarction. Circulation. 103(23):2776-9.
Shyu WC, Lin SZ, Yang HI, Tzeng YS, Pang CY, Yen PS, Li H. (2004) Functional recovery of
stroke rats induced by granulocyte colony-stimulating factor-stimulated stem cells.
Circulation 110(13):1847-54.
Six I, Gasan G, Mura E, Bordet R. (2003) Beneficial effect of pharmacological mobilization of
bone marrow in experimental cerebral ischemia. Eur J Pharmacol. 458(3):327-8.
Snykers S, De Kock J, Tamara V, Rogiers V. (2011) Hepatic differentiation of mesenchymal
stem cells: in vitro strategies. Methods Mol Biol. 698:305-14.
Soonpaa MH and Field LJ. (1998) Survey of studies examining mammalian cardiomyocyte
DNA synthesis. Am Heart Assoc 1998;83:15–26.
Spees JL, Olson SD, Ylostalo J, Lynch PJ, Smith J, Perry A, Peister A, Wang MY, Prockop DJ.
(2003) Differentiation, cell fusion, and nuclear fusion during ex vivo repair of
epithelium by human adult stem cells from bone marrow stroma. Proc Natl Acad
Sci U S A. 100(5):2397-402.
Stamm C, Westphal B, Kleine HD, Petzsch M, Kittner C, Klinge H, Schümichen C, Nienaber
CA, Freund M, Steinhoff G. (2003) Autologous bone-marrow stem-cell
transplantation for myocardial regeneration. Lancet. 361(9351):45-6.
Stout CL, Ashley DW, Morgan JH 3rd, Long GF, Collins JA, Limnios JI, Lochner F,
McCommon G, Hixson D, Black AC Jr, Young HE. (2007) Primitive stem cells
residing in the skeletal muscle of adult pigs are mobilized into the peripheral blood
after trauma. Am Surg. 73(11):1106-10.
Suratt BT, Cool CD, Serls AE, Chen L, Varella-Garcia M, Shpall EJ, Brown KK, Worthen GS.
(2003) Human pulmonary chimerism after hematopoietic stem cell transplantation.
Am J Respir Crit Care Med. 168(3):318-22.
Tissue Regeneration – From Basic Biology to Clinical Application
200
Swenson ES, Kuwahara R, Krause DS, Theise ND. (2008) Physiological variations of stem
cell factor and stromal-derived factor-1 in murine models of liver injury and
regeneration. Liver Int. 28(3):308-18
Takahashi T, Kalka C, Masuda H, Chen D, Silver M, Kearney M, Magner M, Isner JM,
Asahara T. (1999) Ischemia- and cytokine-induced mobilization of bone marrowderived
endothelial progenitor cells for neovascularization. Nat Med. 5(4):434-8.
Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L,
Petersen BE, Scott EW. (2002) Bone marrow cells adopt the phenotype of other cells
by spontaneous cell fusion. Nature. 416(6880):542-5.
Theise ND, Nimmakayalu M, Gardner R, Illei PB, Morgan G, Teperman L, Henegariu O,
Krause DS. (2000) Liver from bone marrow in humans. Hepatology. 32(1):11-6.
Tirino V, Paino F, d’Aquino R, Desiderio V, De Rosa A, Papaccio G. (2011) Methods for the
Identification, Characterization and Banking of Human DPSCs: Current Strategies
and Perspectives. Stem Cell Rev. Feb 12. [Epub ahead of print]
Tomita M, Adachi Y, Yamada H, Takahashi K, Kiuchi K, Oyaizu H, Ikebukuro K, Kaneda H,
Matsumura M, Ikehara S. (2002) Bone marrow-derived stem cells can differentiate
into retinal cells in injured rat retina. Stem Cells. 20(4):279-83.
Tomoda H and Aoki N. (2003) Bone marrow stimulation and left ventricular function in
acute myocardial infarction. Clin. Cardiol. 26:455-457
Trzaska KA, Rameshwar P. (2011) Dopaminergic Neuronal Differentiation Protocol for
Human Mesenchymal Stem Cells. Methods Mol Biol. 698:295-303.
Tsagias N, Koliakos I, Lappa M, Karagiannis V, Koliakos GG. (2011) Placenta perfusion has
hematopoietic and mesenchymal progenitor stem cell potential. Transfusion.
51(5):976-85.
Tse HF, Kwong YL, Chan JK, Lo G, Ho CL, Lau CP. (2003) Angiogenesis in ischaemic
myocardium by intramyocardial autologous bone marrow mononuclear cell
implantation. Lancet. 361(9351):47-9.
Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, Zeiher AM, and Dimmeler
S. (2001) Number and migratory activity of circulating endothelial progenitor cells
inversely correlate with risk factors for coronary artery disease. Circ Res. 89:e1-e7.
Vasania VS, Prasad P, Gill RK, Mehta A, Viswanathan C, Sarang S, Majumdar AS. (2010)
Molecular and cellular characterization of expanded and cryopreserved human
limbal epithelial stem cells reveal unique immunological properties. Exp Eye Res.
92(1):47-56.
Vassilopoulos G, Wang PR, Russell DW. (2003) Transplanted bone marrow regenerates liver
by cell fusion. Nature. 422(6934):901-4.
Viktorov IV, Savchenko EA, Ukhova OV, Alekseyeva NY, Chekhonin VP. (2006)
Multipotent stem and progenitor cells of the olfactory epithelium. Bull Exp Biol
Med. 142(4):495-502.
Voermans C, Rood PM, Hordijk PL, Gerritsen WR, van der Schoot CE. (2000) Adhesion
molecules involved in transendothelial migration of human hematopoietic
progenitor cells. Stem Cells. 18(6):435-43.
Voltarelli JC, Couri CEB, Stracieri ABPL, Oliveira MC, Moraes DA, Pieroni F, Coutinho M,
Malmegrim KCR, Foss-Freitas MC, Simoes BP, Foss MC, Squiers E, and Burt RK.
The Therapeutic Potential of Stimulating Endogenous Stem Cell Mobilization
201
(2007) Autologous nonmyeloablative hematopoietic stem cell transplantation in
newly diagnosed Type 1 diabetes mellitus. JAMA. 297(14):1568-1576.
Wang J, Wang X, Sun Z, Wang X, Yang H, Shi S, Wang S. (2011) Stem cells from humanexfoliated
deciduous teeth can differentiate into dopaminergic neuron-like cells.
Stem Cells Dev. 19(9):1375-83.
Welte K, Platzer E, Lu L, Gabrilove JL, Levi E, Mertelsmann R, and Moore MA. (1985)
Purification and biochemical characterization of human pluripotent hematopoietic
colony-stimulating factor. Proc Natl Acad Sci U S A. 82(5):1526-30.
Werner N, Kosiol S, Schiegl T, Ahlers P, Walenta K, Link A, Böhm M, Nickenig G. (2005)
Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J
Med. 353(10):999-1007.
Westerweel PE, Luijten RK, Hoefer IE, Koomans HA, Derksen RH, Verhaar MC. (2007)
Haematopoietic and endothelial progenitor cells are deficient in quiescent systemic
lupus erythematosus. Ann Rheum Dis. 66(7):865-70.
Willing AE, Vendrame M, Mallery J, Cassady CJ, Davis CD, Sanchez-Ramos J, Sanberg PR.
(2003) Mobilized peripheral blood cells administered intravenously produce
functional recovery in stroke. Cell Transplant.12(4):449-54.
Wojakowski W, Tendera M, Michałowska A, Majka M, Kucia M, Maślankiewicz K, Wyderka
R, Ochała A, Ratajczak MZ. (2004) Mobilization of CD34/CXCR4+, CD34/CD117+,
c-met+ stem cells, and mononuclear cells expressing early cardiac, muscle, and
endothelial markers into peripheral blood in patients with acute myocardial
infarction. Circulation. 110(20):3213-20.
Wojakowski W, Tendera M, Zebzda A, Michalowska A, Majka M, Kucia M, Maslankiewicz
K, Wyderka R, Król M, Ochala A, Kozakiewicz K, Ratajczak MZ. (2006)
Mobilization of CD34(+), CD117(+), CXCR4(+), c-met(+) stem cells is correlated
with left ventricular ejection fraction and plasma NT-proBNP levels in patients
with acute myocardial infarction. Eur Heart J. 27(3):283-9.
Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Breidenbach C, Fichtner S,
Korte T, Hornig B, Messinger D, Arseniev L, Hertenstein B, Ganser A, Drexler H.
(2004) Intracoronary autologous bone-marrow cell transfer after myocardial
infarction: the BOOST randomised controlled clinical trial. Lancet. 364(9429):141-8.
Woodbury D, Schwarz EJ, Prockop DJ and Black IB. (2000) Adult rat and human bone
marrow stromal cells differentiate into neurons. J. Neurosci. Res. 61, 364-370.
Wright DE, Bowman EP, Wagers AJ, Butcher EC, Weissman IL. (2002) Hematopoietic stem
cells are uniquely selective in their migratory response to chemokines. J Exp Med.
195(9):1145-54.
Wurmser AE, Gage FH. (2002) Stem cells: cell fusion causes confusion. Nature.
416(6880):485-7.
Yamamoto S, N Yamamoto, T Kitamura and M Nakafuku. (2001). Proliferation of
parenchymal neural progenitors in response to injury in the adult rat spinal cord.
Exp Neurol 172:1115–1127.
Yoo SW, Kim SS, Lee SY, Lee HS, Kim HS, Lee YD, Suh-Kim H. (2008) Mesenchymal stem
cells promote proliferation of endogenous neural stem cells and survival of
newborn cells in a rat stroke model. Exp Mol Med. 40(4):387-97.
Tissue Regeneration – From Basic Biology to Clinical Application
202
Zachar V, Rasmussen JG, Fink T. (2011) Isolation and growth of adipose tissue-derived stem
cells. Methods Mol Biol. 698:37-49.
Zhang CP, Fu XB. (2008) Therapeutic potential of stem cells in skin repair and regeneration.
Chin J Traumatol. 11(4):209-21.
Zhang X, Hirai M, Cantero S, Ciubotariu R, Dobrila L, Hirsh A, Igura K, Satoh H, Yokomi I,
Nishimura T, Yamaguchi S, Yoshimura K, Rubinstein P, Takahashi TA. (2011)
Isolation and characterization of mesenchymal stem cells from human umbilical
cord blood: reevaluation of critical factors for successful isolation and high ability
to proliferate and differentiate to chondrocytes as compared to mesenchymal stem
cells from bone marrow and adipose tissue. J Cell Biochem. 112(4):1206-18.
Zhu J, Wang X, Chen J, Sun J, Zhang F. (2006) Reduced number and activity of circulating
endothelial progenitor cells from patients with hyperhomocysteinemia. Arch Med
Res. 37(4):484-9.
Zohlnhöfer D, Dibra A, Koppara T, de Waha A, Ripa RS, Kastrup J, Valgimigli M, Schömig
A, Kastrati A. (2008) Stem cell mobilization by granulocyte colony-stimulating
factor for myocardial recovery after acute myocardial infarction: a meta-analysis. J
Am Coll Cardiol. 51(15):1429-37.
Zou Y, Kottmann AH, Kuroda M, Taniuchi I, Littman DR. (1998) Function of the chemokine
receptor CXCR4 in haematopoiesis and in cerebellar development. Nature 393:595–
599