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Stem cells
STEM CELLS
Stem Cells’ Definition:
Stem cells are defined as ‘immature’ or undifferentiated cells which are
found in multicellular organisms. They are able to produce identical daughter cells
(clonogenic), divide indefinitely (self-renewing), and differentiate into multiple cell
lineages (potent) giving different cell types with different functions (Robey, 2000).
Stem cells remain uncommitted until they receive a signal to develop into
specialized cell. They may remain dormant or quiescent for prolonged periods until
they are exposed to a particular stimulus. A stem cell is able to produce at least one
and often many types of highly-differentiated cell. They serve as a repair system
being able to divide without limit to replenish other cells. When stem cells divide,
each new cell has the potential to either remain as a stem cell or become another
cell type with new special functions, such as blood cells, nerve cells, or heart cells
(Little et al., 2006).
Stem Cells Properties:
Stems cells are those that have 3 general properties:
(1) They are capable of dividing and renewing themselves for long periods (selfrenewal).
(2) They are unspecialized.
(3 They can differentiate into multiple types of functionally mature specialized
cells (Multipotency) (Blau et al., 2001).
Stem cells
(1)
Self-renewal:
Stem cells have the capacity for self-renewal (undergoing cycles of mitotic
division while maintaining the same undifferentiated state), which ensures that
stem cell reserves are not exhausted throughout time (He et al., 2009).
For stem
cells to maintain themselves in tissue and at the same time provide cells to
maintain the differentiated function of that tissue, stem cells fate is controlled by
two general modes of cell division. The first mechanism is asymmetric cell
division such that it produces one differentiated daughter cell and one cell that is
still a stem cell. This allows maintaining a constant number of stem cells, which is
generally sufficient under physiological conditions. The second mechanism,
symmetric cell division is a highly regulated process in which a stem cell gives rise
to daughter cells that have a finite probability of either becoming stem cells or
differentiated cells. This leads to an expansion of the stem cell pool, a condition
required after tissue injury or in diseased conditions causing loss of differentiated
cells (Yu et al., 2006).
The mechanisms that regulate these division processes are complex and may be
a combination of both mechanisms depending on the cellular environment or
biological needs (Pattern et al., 2000).
(2)
Stem cell potency:
The ability to differentiate into specialized cell types and be able to give rise to
any mature cell type is referred to as potency. Potency of the stem cell specifies
the differentiation potential i.e., the potential to differentiate into different cell
types. According to potency stem cells can be classified into:
pluripotent and multipotent (Hima Bindu & Srilath, 2011).
totipotent,
Stem cells
 Totipotent stem cells:
They are cells that can generate full functional organism, as well as the placenta
and other supporting tissues (embryonic as well as extra-embryonic tissues).
The only totipotent cells are the fertilized egg and the cells produced by the first
few divisions of the fertilized egg as they can give rise to any type of cell: cells of
the trophoblast and cells of the 3 germ layers (endoderm, mesoderm, and
ectoderm), all of which are necessary for complete embryonic development
(Sorapop et al., 2006).
 Pluripotent stem cells:
They are the descendants of totipotent cells and can differentiate into nearly all
cells, i.e. cells derived from any of the three embryonic germ layers (ectoderm,
mesoderm and endoderm) but not the whole organism (can not give rise to
trophoblasts). They are classified into: embryonic stem cells (ESCs), embryonic
germ cells, embryonic carcinoma cells and multipotent adult progenitor cell from
bone marrow (Muller-Sieburg et al., 2002).
 Multipotent stem cells:
Stem cells that have a more retricted ability to give rise to cells of different lineages
within a single germ layer are considered multipotent (adult stem cells) .They can
differentiate into a number of cells, but only those of a closely related family of
cells. These are true stem cells but can only differentiate into a limited number of
types. They include haemopoietic stem cells, neuronal stem cells and mesenchymal
stem cells (Krause et al., 2001).
Stem cells
 Unipotent stem cells:
Tissue-specific progenitor stem cells or unipotent stem cells can differentiate
and give rise to only one cell type, their own. Unipotentiality of cells means, only
able to generate one cell type but have the property of self-renewal, which
distinguishes them from non-stem cells. Unipotent stem cells include the stem cells
in the gut epithelium, the skin, and the seminiferous epithelium of the testis. Most
epithelial tissues self-renew throughout adult life due to the presence of unipotent
progenitor cells (Blanpain et al., 2007).
Induced pluripotent stem cells:
Induced pluripotent stem cells (iPSCs) are a type of pluripotent stem cells that
can be produced from adult somatic cells by genetic reprograming or forced
introduction of genes. iPSCs are epigenetically reprogrammed to lose tissuespecific features and gain pluripotency. Similar to hESCs, they can theoretically
differentiate into any type of cells ( Patel & Yang, 2010).
In 2006, Takahashi and Yamanaka introduced the concept of induced
pluripotent stem cells by generating stem cells by using a combination of 4
reprogramming factors, including Oct4 (Octamer binding transcription factor-4),
Sox2 (Sex determining region Y)-box 2, Klf4 (Kruppel Like Factor-4), and c-Myc
and were demonstrated both self-renewing and differentiating like ESCs
(Takahashi & Yamanaka, 2006). In 2007, they reported that a similar approach
was applicable for human fibroblasts and human iPSCs can be generated
(Takahashi et al., 2007). James Thomson's group also reported the generation of
human iPSC using a different combination of factors. Since then, a number of
different reprogramming factors and methods have been established (Yu et al.,
2007).
Stem cells
Initially iPSCs were generated using either retroviruses or lentiviruses, which
might cause insertional mutagenesis and thus could perhaps even lead to adverse
effects like those seen in some attempts at gene therapy (Hacein-Bey-Abina et al.,
2003). In addition, retroviruses may make iPSCs immunogenic (Zhao et al., 2011).
Thus, for the purpose of cell transplantation therapy many ways to generate
integration-free iPSCs have been reported. These methods include plasmid (Okita
et al., 2008), adenovirus (Stadtfeld et al., 2008), synthesized RNAs (Warren et al.,
2010), and proteins (Kim et al., 2009).
Figure (1): Reprogramming of adult stem cells into iPS cells (Meregalli et al.,
2011).
Patient-derived iPSCs have been shown to be useful tools for drug
development and modeling of diseases. Scientists hope to use them in
transplantation medicine. Patient-specific iPSCs can be used to recapitulate
phenotypes of not only monogenic diseases but also late-onset polygenic diseases,
such as Parkinson's disease (Devine et al., 2011), Alzheimer's disease (Israel et al.,
Stem cells
2012), and schizophrenia (Brennand et al., 2011). Excitement surrounds the
potential for application of these cells to both analysis of disease mechanisms and
investigation of potential new treatments. Somatic cells derived from iPSCs,
particularly cardiac myocytes and hepatocytes, could also be used for toxicology
testing as an alternative to existing approaches (Yamanaka, 2009).
In addition, iPSCs can be used in animal biotechnology and genetic
engineering. This allows for the generation of disease models and the production of
useful substances, such as enzymes, that are deficient in patients with genetic
diseases. One of the most striking applications of iPSCs was reported by Nakauchi
and coworkers, who generated a rat pancreas in a mouse, by microinjecting rat
iPSCs into mouse blastocysts deficient in a gene essential for pancreas
development. In the future, it might become possible to generate organs for human
transplantation using a similar strategy (Kobayashi et al., 2010).
Stem Cells Origin:
(1)
Embryonic Stem Cells (ESC):
Embryonic stem cells derived from the inner cell mass of the blastocyst (5- to 7day-old embryo) possess two important characteristics: self-renewal and
pluripotency. ES cell lines are sometimes referred to as immortal due to their
ability to keep dividing or self-renewing over many generations. Derivation of
human pluripotent embryonic stem cell lines and recent advances in hESC biology
has created great interest in the field of stem cell-based engineering, but there is
Stem cells
ethical
debates
surrounding
their
use
(Rippon
&
Bishop,
2004).
Figure (2): Differentiation potentiality of human embryonic stem cell lines
(Meregalli et al., 2011).
(2)
Fetal Stem Cells:
Fetal stem cells are primitive cell types found in the organs of fetus
(approximately 10 weeks of gestation). Fetal stem cells can be isolated from
fetal blood and bone marrow as well as from other fetal tissues, including liver
and kidney. Fetal stem cells are generally tissue-specific, generating the mature
cell types within the particular tissue or organ they are found in. This type of
stem cell is currently grouped into an adult stem cell (Guillot et al., 2006).
Stem cells
(3)
Cord Blood Stem Cells:
Blood from the umbilical cord contains multipotent stem cells that are
genetically identical to the newborn. The umbilical cord blood is often banked,
or stored, for possible future use of stem cell therapy (Lee et al., 2004).
(4)
Adult Stem Cells:
Adult stem cells are undifferentiated cells that occur in a developed
organism in the post natal state and have two properties: the ability to divide and
renew themselves and to be specialized to give cell types of the tissue of origin.
Adult stem cells are lineage-restricted (multipotent) and are generally referred to
by their tissue origin. They are found both in children, as well as adults. Their
main role is thought to be maintenance and repair of tissues (Tuan et al., 2003).
Adult stem cells are classified by the source tissue or types of cells from
which they are derived into: bone marrow stem cells, peripheral blood stem
cells, heart and lung stem cells, intestinal and liver stem cells, pancreatic stem
cells, kidney stem cells, central nervous system stem cells, skin stem cells,
skeletal muscle stem cells, dental pulp derived stem cells, and adipose tissue
stem cells (Toma et al., 2001).
Bone marrow stem cells:
The bone marrow (BM) contains three kinds of stem cells: endothelial stem
cells, hematopoietic stem cells and mesenchymal stem cells (bone marrow
stromal cells).
1- Endothelial stem cells: Endothelial progenitor cells (EPCs) are present in BM
and blood. They have the ability to differentiate into endothelial cells, the cells
Stem cells
that line the inner surfaces of blood vessels throughout the body. They
participate in new vessel formation (neovascularization) in physiological states
as wound healing and in pathological states as tumor angiogenesis (Folkman,
1998).
2- Hematopoietic stem cells (HSCs):
HSCs are present in bone marrow,
peripheral blood and umbilical cord blood. They give rise to all the types of
blood cells through the process of haematopoiesis. They give rise to both the
myeloid and lymphoid lineages of blood cells. Myeloid cells include monocytes,
macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells,
and megakaryocytes or platelets. Lymphoid cells include T cells, B cells, and
natural killer cells (Kondo et al., 2003).
3- Mesenchymal stem cells: MSC refer to a postnatal, multipotent and
self-renewing precursor derived from an original embryonic MSC. They
function to maintain the turnover of skeletal tissues in homeostasis or tissue
repair during adulthood. MSCs progress through discrete stages of
differentiation in an orderly manner to give rise to functionally and
phenotypically mature tissues, including bone, smooth muscle, tendons and
cartilage (Bianco et al., 2001).
Stem cells
Stem Cell Niche:
Stem cell niche is defined as the cellular and molecular microenvironments that
regulate stem cell function, provides signals to stem cells in the form of secreted
and cell surface molecules to control the rate of stem cell proliferation, determine
the fate of stem cell daughters, and protect stem cells from exhaustion or death.
This includes control of the balance between quiescence, self-renewal, and
differentiation, as well as the engagement of specific programs in response to stress
(Xie & Spradling, 2000).
Within the human body, stem-cell niches maintain adult stem cells in a
quiescent state, but after tissue injury, the surrounding micro-environment actively
signals to stem cells to promote either self-renewal or differentiation to form new
tissues. Several factors are important to regulate stem-cell characteristics within the
niche: cell–cell interactions between stem cells, as well as interactions between
stem cells and neighbouring differentiated cells, interactions between stem cells
and adhesion molecules, the oxygen tension, extracellular matrix components,
cytokines, growth factors and the physicochemical nature of the environment
including pH, ionic strength and metabolites (O’Brien & Bilder 2013).
Stem cells
Figure (3): Composition of the niche. Stem cell niches are complex, heterotypic,
dynamic structures, which include different cellular components, secreted factors, immunological
control, ECM, physical parameters and metabolic control (Lane et al., 2014).
Stem cells
Mesenchymal Stem Cells:
1- Defining mesenchymal stem cells:
Stem cells are classically defined by their multipotency and selfrenewal. They
are derived from mesodermal germ layer. Their function is to maintain the turnover
of skeletal tissues in homeostasis or tissue repair during adulthood (Chanda et al.,
2010).
MSCs were firstly identified in the 1960s as colony-forming unit fibroblasts
(CFU-Fs) by Friedenstein and coworkers who observed that the bone marrow is a
source of stem cells for mesenchymal tissue. They described these cells as
mononuclear nonphagocytic cells with fibroblast-like phenotype and colongenic
potential capable of adhering to the culture surface in a monolayer culture
(Friedenstein et al., 1968). Friedenstein was the first investigator to demonstrate
the ability of MSCs to differentiate into mesoderm-derived tissue. During the
1980s, MSCs were shown to differentiate into osteoblasts, chondrocytes, and
adipocytes (Piersma et al., 1985). In the 1990s, MSCs were shown to differentiate
into a myogenic phenotype (Wakitani et al., 1995). In 1999, Kopen et al. described
the capacity of MSCs to transdifferentiate into ectoderm-derived tissue.
2- Characterization of Mesenchymal Stem Cells:
MSCs express a large number of adhesion molecules, extracellular matrix
proteins, cytokine and growth factor receptors, associated with their function and
cell interactions within the bone marrow stroma (Deans and& Moseley, 2000).
The International Society for Cell Therapy proposed criteria for MSCs that
comprise: (1) adherence to plastic in standard culture conditions; (2) expression of
Stem cells
the surface molecules CD73, CD90, and CD105 in the absence of CD34 (the
primitive hematopoietic stem cell marker), CD45 (a marker of all hematopoietic
cells), HLA-DR, CD14 or CD11b (an immune cell marker), CD79a, or CD19
surface molecules as assessed by fluorescence-activated cell sorter analysis; and
(3) a capacity for differentiation into mesodermal cells such as osteoblasts, adipose
cells, cartilage cells or skeletal muscle cells under standard differentiation
conditions in vitro (Dominici et al, 2006). The typical CD markers of bone
marrow-derived MSCs are, CD105, CD73CD90, CD45−, CD34− and HLA-DR−
(Hagmann et al., 2013).
These criteria were established to standardize human MSC isolation but may
not apply uniformly to other species. For example, murine MSCs have been shown
to differ in behavior and marker expression when compared with human MSCs. It
is believed that certain in vivo surface markers may no longer be expressed after
explantation when MSCs are isolated and expanded in culture, although new
markers are acquired during expansion (Peister et al., 2004).
MSCs Multilineage Differentiation Potential:
Differentiation of MSCs is regulated by genetic events, involving transcription
factors. Differentiation to a particular phenotype pathway can be controlled by
some regulatory genes which can induce progenitor cells’ differentiation to a
specific lineage. The microenvironment has the strongest influence on the
maturation and differentiation of MSCs. However, growth factors, induction
chemicals, cell-to-cell communication, physical factors and cell structure were
found to have an effect (Ding et al., 2011).
Stem cells
1. Mesoderm Differentiation: Theoretically, mesodermal differentiation is
easily attainable for MSCs because they are from same embryonic origin. For
osteogenesis, MSCs in the presence of dexamethasone, 𝛽-glycerophosphate,
and ascorbic acid express alkaline phosphatase and calcium accumulation, a
morphology
consistent
with
osteogenic
differentiation.
Osteogenic
differentiation of MSCs is a complex process controlled by multiple signaling
pathways and transcription factors. Runt related transcription factor 2 (Runx2)
and Caveolin-1 are considered key regulators of osteogenic differentiation.
Bone morphogenetic proteins (BMPs), especially BMP-2, BMP-6, and BMP-9,
strongly promote osteogenesis in MSCs. BMP-2 induces the p300 mediated
acetylation of Runx2, a master osteogenic gene, which results in enhanced
Runx2 transactivating capability. The acetylation is specific to histone
deacetylases 4 and 5, which, by deacetylating Runx2, promote its subsequent
degradation by Smurf1 and Smurf2, and E3 ubiquitin ligases (Hwang et al.,
2009). Core binding factor alpha-1/osteoblast-specific factor2 (cbfa1/osf2) and
Wnt signaling are also involved in osteogenic differentiation of MSCs (Gaur et
al., 2005).
In adipogenesis, dexamethasone and isobutyl-methylxanthine (IBMX) and
indomethacin (IM) have been used for induction of differentiation. Peroxisome
proliferator-activated receptors-𝛾2 (PPAR𝛾2), CCAAT/enhancer binding
protein (C/EBP), and retinoic C receptor have been implicated in adipogenesis.
Phosphatidylinositol 3-kinase (PI3K) activated by Epac leads to the activation
of protein kinase B (PKB)/cAMP response element-binding protein (CREB)
signaling and the upregulation of PPAR𝛾 expression, which in turn activate the
transcription of adipogenicgenes (Ding et al., 2011).
Stem cells
In chondrogenesis, transforming growth factor (TGF)-𝛽1 and 𝛽2 are
reported to be involved. Differentiation of MSCs into cartilage is characterized
by upregulation of cartilage specific genes, collagen type II, IX, aggrecan, and
biosynthesis of collagen and proteoglycans. The emerging results suggested the
possible roles of Wnt/𝛽catenin in determining differentiation commitment of
mesenchymal cells between osteogenesis and chondrogenesis (Huang et al.,
2004). A recent report suggested that miR-449a regulates the chondrogenesis of
human MSCs through direct targeting of Lymphoid Enhancer-Binding Factor-1
(Paik et al., 2012). Elevated 𝛽catenin signaling induces Runx2, resulting in
osteoblast differentiation, whereas reduced 𝛽-catenin signaling has the opposite
effect on gene expression, inducing chondrogenesis (Day et al., 2005).
Figure (4): Molecular regulation of MSC differentiation. Wnt signaling and TGF𝛽
induce intracellular signaling and regulate differentiation of MSCs (Williams & Hare, 2011).
Stem cells
2. Ectoderm Differentiation: In vitro neuronal differentiation of MSCs can be
induced by DMSO, butylated hydroxyanisole (BHA), 𝛽-mercaptoethanol, KCL,
forskolin, and hydrocortisone (Ding et al., 2011). Moreover, Notch-1 and protein
kinase A (PKA) pathways are found to be involved in neuronal differentiation. In
presence of other stimulatory factors, downregulation of caveolin-1 promotes the
neuronal differentiation of MSCs by modulating the Notch signaling pathway
(Wang et al., 2012).
3. Endoderm Differentiation: In liver differentiation, hepatocyte growth factor
and oncostatin M were used for induction to obtain cuboid cells which expressed
appropriate
markers
(𝛼-fetoprotein,
glucose
6-phosphatase,
tyrosine
aminotransferase, and CK-18) and albumin production in vitro. Recent studies
identified methods to develop pancreatic islet 𝛽-cell differentiation from adult stem
cells. The resulting cells showed specific features, high insulin-1 mRNA content,
and synthesis of insulin and nestin (Bhandari et al., 2010). Murine adipose tissuederived mesenchymal stem cells can also differentiate to endoderm islet cells
(expressing Sox17, Foxa2, GATA-4, and CK-19) with high efficiency then to
pancreatic endoderm (Pancreatic and duodenal homeobox 1[Pdx-1], Ngn2,
Neurogenic differentiation [NeuroD], paired box-4 [PAX4], and Glut-2), and
finally to pancreatic hormone-expressing (insulin, glucagon, and somatostatin)
cells (Chandra et al., 2009).
Stem cells
Figure (4): MSCs multilineage differentiation potential. Adapted from Caplan &
Bruder (2001).
Stem cells
3-
Sources of Mesenchymal Stem Cells:
MSCs can be derived from many tissue sources: BM, adipose tissue, synovial
tissue, lung tissue, umbilical cord blood, and peripheral blood. Despite sharing
similar characteristics, these MSCs from different sources differ in their
differentiation potential and gene expression profile (Baksh et al., 2007).
MSCs have been isolated from nearly every tissue type (brain, spleen, liver,
kidney, lung, BM, muscle, thymus, aorta, vena cava, pancreas) of adult mice, which
suggests that MSCs may reside in all postnatal organs (da Silva et al., 2006).
MSCs have been reported to constitute about 0.01%–0.001% of the bone
marrow mononuclear population. Stem cells harbored in the bone marrow are
considered to have the highest multilineage potential. These cells can be isolated
from marrow aspirates of the superior iliac crest, femur, and tibia. For this purpose,
marrow cells are usually enriched for mononuclear cells with Ficoll or Percol and
then plated on culture plastic vessels in order to prepare adherent cell populations.
It has recently been demonstrated that late plastic adherent MSCs possess higher
osteogenic potential (Eslaminejad et al., 2006).
Adipose tissue as well as birth-associated tissues, including umbilical cord and
dental pulp has been found to contain MSC-like population. The presence of cells
with multipotent diff erentiation capacity in adipose tissue is promising due to the
ease of accessibility of adipose tissue and its abundance in the body. Adipose tissue
can be an appropriate substitute for marrow in regenerative medicine and tissue
engineering . Adipose-derived stromal cells (ADSCs) can be derived from adipose
collected by liposuction and lipectomy. ADSCs are able to maintain proliferation
potential as well as diff erentiation capacity even in older people. By now, many
studies conducted on animal models have confirmed the regenerative potential of
ADSCs in bone defects (Bunnell et al., 2008).
Stem cells
The umbilical cord from a newborn baby contains two arteries and a vein
covered with mucus connective tissue rich in hyaluronic acid, referred to as
Wharton’s jelly. According to studies, MSC-like cells can be derived from various
components of this cord. For example, blood from an umbilical cord is a rich
source for pluripotent cells referred to as umbilical-cord-blood-derived MSCs
(UCB-MSCs). These cells are quite similar to marrow derived MSCs and have
osteogenic potential in an optimized culture (Hutson et al., 2005).
Several stem cell types in dental tissue have been reported including dental
pulp stem cells (DPSCs), stem cells from human exfoliated deciduous teeth
(SHED), stem cells of the apical papilla (SCAP), periodontal ligament stem cells
(PDLSCs), and dental follicle progenitor cells (DFPCs) (Nakamura et al., 2009).
4- Immunology of Mesenchymal Stem Cells:
Human MSCs express moderate levels of human leukocyte antigen (HLA)
major histocompatibility complex class I, lack major histocompatibility complex
class II expression, and do not express costimulatory molecules B7 and CD40
ligand (Tse et al., 2003). Tolerance of MSCs as an allogeneic transplant is due to
this unique immunophenotype coupled with powerful immunosuppressive activity
via cell-cell contact with target immune cells and secretion of soluble factors, such
as nitric oxide, indoleamine 2,3-dioxygnease, and heme oxygenase1. MSCs
produce an immunomodulatory effect by interacting with both innate and adaptive
immune cells (Figure 5) (Ren et al., 2008).
The innate immune cells (neutrophils, dendritic cells, natural killer cells,
eosinophils, mast cells, and macrophages) are responsible for a nonspecific defense
to infection, and MSCs have been shown to suppress most of these inflammatory
cells. Neutrophils are one of the first cells to respond to an infection, and an
Stem cells
important process in their response to inflammatory mediators is the respiratory
burst, characterized by large oxygen consumption and production of reactive
oxygen species. MSCs have shown to dampen the respiratory burst process by
releasing interleukin (IL)-6. MSCs have been shown to inhibit the differentiation
of immature monocytes into dendritic cells which play an important role in antigen
presentation to naïve T cells. Additionally, cocultures of MSCs and dendritic cells
inhibit the production of tumor necrosis factor- 𝛼, a potent inflammatory molecule
(Aggarwal & Pittenger, 2005).
Natural killer cells are important innate cells in the defense against viral organisms
and in tumor defense by their secretion of cytokines and cytolysis. MSCs cultured
with freshly isolated resting natural killer cells have been shown to inhibit IL-2–
induced proliferation and decrease secretion of interferon (IFN- 𝛾) by 80%.
However, IL-2–activated natural killer cells can lyse autologous and allogeneic
MSCs. The adaptive immune system, composed of T and B lymphocytes, is
capable of generating specific immune responses to pathogens with the production
of memory cells. Once a T cell is activated by a foreign antigen binding to a
specific T-cell receptor, the T cell proliferates and releases cytokines (Uccelli et
al., 2008).
T cells exist as CD8+ cytotoxic T cells that induce death of target cells or CD4 +
helper T cells that modulate the other immune cells. MSCs have been shown to
suppress T-cell proliferation in a mixed lymphocyte culture. Proliferation of
cytotoxic and helper T cells are suppressed via soluble factors released by MSCs,
such as hepatocyte growth factor (HGF) and transforming growth factor (TGF) 𝛽1.
When MSCs are present during naïve T-cell differentiation to CD4+ T-helper cells,
there is a marked decrease in the production of interferon and an increase in the
production of IL-4. MSCs are suggested to alter naïve T cells from a
Stem cells
proinflammatory state (heavy production of interferon- ) to an anti-inflammatory
state (greater production of IL-4) (Aggarwal & Pittenger, 2005).
It has been demonstrated that MSCs stop the B-cell cycle at the G0/G1 stage
and inhibit their diff erentiation into plasma cells (Corcione et al., 2006).
Ramasamy et al. (2007) have indicated that BMSCs are able to inhibit dendritic
cell (DC) diff erentiation and prevent them from entering into the cell cycle. DCs
are able to efficiently present antigens to lymphocytes.
Figure (5): MSC interactions with immune cells. MSCs are immunoprivileged cells
that inhibit both innate (neutrophils, dendritic cells, natural killer cells) and adaptive (T cells
and B cells) immune cells. INF, interferon; TNF, tumor necrosis factor. (Williams & Hare,
2011).
Stem cells
5- Delivery of mesenchymal stem cells:
The routes of MSC administration are classified into two categories: systemic
and topical. There are two types of topical administration: intralesional injection
(e.g., intracranial, intracerebral, and subcutaneous) and local vascular injection
(e.g., superior vena cava, mesenteric blood vessels, coronary artery). Compared
with systemic administration, topical administration routes may have a common
advantage in that MSCs arrive directly at the target tissue with little loss during
migration (Kim et al., 2014).
Types of systemic administration include intravenous (IV), intraperitoneal (IP),
intra-arterial (IA) and intracardiac (IC). IV is the most common method in
preclinical and clinical settings because of its convenience. However, MSCs
administered via this route are more easily trapped in small lung capillaries
because of their larger size and expression of cell adhesion molecules. Lung
entrapment of MSCs decreases the number of MSCs delivered to target tissues and
can result in ineffectual treatment. However, some reports have shown that MSCs
delivered via IV injection have protective effects in various animal models even
when lung entrapment occurs (Makela et al., 2015).
Administration routes determine the microenvironments that MSCs first
encounter after entering the patient’s body, thus influencing their differentiation,
immunogenicity, and survival (Ishikane et al., 2010).
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6- Migration and Homing of Mesenchymal Stem Cells:
One of the most important features of MSCs is their ability to reach
damaged
tissue
in
response
to
a
correct
combination
of
signaling
molecules from the injured tissue and corresponding receptors on the
MSCs themselves. Therapeutic efficacy of MSC depends on efficient
mobilization from their bone marrow niches and trafficking through the
circulation to the injured or stressed tissue (Ponte et al., 2007).
At the injury site, MSCs could possibly help with repair in two ways:
(1) they diff erentiate to tissue cells in order to restore lost morphology as
well as function, and (2) MSCs secrete a wide spectrum of bioactive factors
that help to create a repair environment by possessing antiapoptotic eff ects,
immunoregulatory function, and the stimulation of endothelial progenitor
cell proliferation (Granero-Molt´o et al., 2009).
Factors Regulating MSC Homing to Damaged or Tumor Tissues:

Oxygen Level: Hypoxic tissues express genes that increase cell
survival under hypoxic conditions and re-establish the vasculature for
oxygen
delivery
production
of
(Semenza,
chemotactic
2000).
factors
In
addition,
implicated
hypoxia
in
cell
induces
the
migration,
differentiation and new bone formation. The platelets, inflammatory cells
and macrophages arriving at the site of injury secrete cytokines and growth
factors, including IL-1 to IL-6, platelet-derived growth factor (PDGF),
vascular endothelial growth factor (VEGF) and bone morphogenetic protein
(BMP). This cellular response leads to the invasion of MSCs, which
differentiate into tissue specific cells to complete the repair (Rui et al.,
2012).
Stem cells
 Chemokines (Chemotactic Cytokines): Chemokines are small proteins
(8–10
KDa)
with
a
capacity
appropriate for the migration
for
creating
a
chemical
environment
of lymphocytes, neutrophils, and
other
immune cells towards inflammation, angiogenesis, and the organogenesis
site. They include:
1- Stromal
factors
cell-derived
is
stimulating
stromal
factor-1
(SDF-1):
cell-derived
factor
factor/CXC
ligand
12
One
1
of
the
best-investigated
(SDF-1)/pre-B
(CXCL12),
cell
considered
a
growthmaster
regulator of CXC receptor 4 (CXCR4)-positive stem and progenitor cells.
SDF-1 signals through its G
CXC
chemokine
receptor-4
protein-coupled trans-membrane receptor
(CXCR4).
When
transfected
at
sites
of
ischemic injury, this factor modulates cell differentiation into mature
reparative cells. SDF-1/CXCR4 signalling is a key factor in bone marrow
stromal cell migration. Therefore, improving the CXCR4 ligand in bone
marrow stromal cells should promote their proliferation and migration
(Wynn et al., 2004).
SDF-1 and other cytokines associate and interact. Shinohara et al
demonstrated that SDF-1 and monocyte chemo-attractant protein 3 (MCP3) jointly regulate the homing of MSCs from the systemic circulation in
fracture repair. The proportion of cells expressing SDF-1 and MCP-3 was
significantly greater in transduced than non-transduced MSCs. Therefore,
the homing of MSCs from the systemic circulation is involved in fracture
repair via an SDF-1–MCP-3 pathway. The two factors act synergistically.
Another study found that by promoting the expression of BMP-2, SDF-1
Stem cells
increased MSC migration and differentiation in promoting fracture healing
(Granero-Moltó et al., 2009).
SDF-1
signaling
is
important
for
maintaining
postnatal
tissue
homeostasis, such as cellular inflammatory and immune response, blood
homeostasis, and bone remodeling (Yu et al., 2003). At the level of cell
function,
the
rearrangements
binding
and
of
SDF-1
integrin
to
activation,
CXCR4
leads
eventually
to
cytoskeleton
resulting
in
the
directional migration of CXCR4-expressing cells towards high gradients
of SDF-1 (Dar et al., 2006).
2-
Transforming
chemotactic
factor
growth
for
factor
bone
β
(TGF-β):
marrow-derived
TGF-β
MSCs.
is
It
an
effective
promotes
the
proliferation of MSCs, preosteoblasts, chondrocytes and osteoblasts and
induces
collagen,
osteopontin,
osteonectin,
proteoglycans,
alkaline
phosphatase and other extracellular proteins (Mendelson et al., 2011).
3-Platelet derived growth factor: Blood PDGF is strongly chemotactic for
inflammatory cells and has a strong stimulatory effect on MSCs and
osteoblast proliferation and migration. From the early to middle stages of
bone healing,
PDGF promotes
the effects
of mesenchymal
cells
in
cartilage and bone formation in development. The combined application of
PDGF and BMP accelerates bone defect repair, but it is still uncertain
whether PDGF can be used for the clinical treatment of fractures (Kodama
et al., 2012).
 Vasculogenesis: It is an important aspect of tissue repair (Kumar et al,
2010). VEGF stimulates MSC mobilization and recruitment to ischemic or
damaged tissues although MSCs do not express receptors for VEGF. In
Stem cells
2007, Ball et al reported VEGF-A acts through platelet derived growth
factor (PDGF) receptors, which determines the vascular cell fate of MSC.
Although chemokine signaling initiates homing of MSCs, it still requires
multiple steps for their passage through the blood vessel and extravasate at
the site of injury or inflammation. Evidence suggests that the MSCs follow
somewhat similar mechanisms utilized by the immune cells to migrate to
inflammatory
sites.
Endothelial
transmigration
is
mediated
by
active
interactions between the molecules (glycoprotein or glycolipids, PSGL-1, Lselectin, integrins, GPCR, LFA-1, Mac-1) expressed on leukocyte surface
and their receptors (E-selectin, P-selectin, PNAd, MAdCAM, VCAM-1,
ICAMs) on the endothelium. Once at the site of tissue damage MSCs have
to extravasate (diapedesis), which involves transient disassembly of the
endothelial
junctions
and
penetration
through
the
underlying
basement
membrane (Luster et al, 2005).
 Expression of Receptors and Adhesion Molecules: Homing is in a significant part
dependent on the chemokine receptor, CXCR4, and its binding partner, stromalderived factor-1 CXCL12. Wynn et al demonstrated that CXCR4 is resent on a
subpopulation of MSCs, which aid in CXCL12-dependent migration and homing.
Aside from CXCR4, freshly isolated BM MSCs and cultured MSCs also express
CCR1, CCR4, CCR7, CCR10, CCR9, CXCR5, and CXCR6 which are also involved
in MSC migration (Honczarenko et al., 2006).
Integrins are another family of cell surface molecules involved in migration of
variety of cells and are expressed on adipose-derived MSC-like cells. integrin
ligands such as VCAM and ICAM are also expressed on MSCs (Krampera et al.,
2006).
Stem cells
 Culture Conditions of MSCs
1-Passage number: It has been shown that with higher passage number, the
engraftment efficiency of MSCs decreased. Rombouts et al. had performed a time
course experiment; they showed that freshly isolated MSCs had a better efficiency of
homing compared to cultured cells (Rombouts & Ploemacher, 2003).
2- Homing receptors:The CXCR4 chemokine receptor that recognizes SDF-1α is
highly expressed on bone marrow MSCs, but is lost upon culturing. However, when
MSCs are cultured with cytokines (such as HGF, SCF, IL-3, and IL-6), and under
hypoxic conditions, CXCR4 expression can be reestablished (Shi et al., 2007).
3-Culture confluence: Matrix metalloproteases (MMPs have been demonstrated to
play a role in MSC migration. Expression of MMPs in MSCs is influenced by
factors such as hypoxia and increased culture confluence (De Becker et al., 2007).
7- Biological properties supporting clinical use of mesenchymal
stem cells in therapy:
MSCs have been known as promising tool for therapeutic purpose based on their
several
advantages
including
selfrenewal,
extensive
in
vitro
expansion,
immunomodulation property, engraftment capacity, multi-lineages differentiation
potential including few ethical concerns as compared to embryonic stem cells.
Moreover, increasing evidences have been shown that MSCs can be isolated from
various cell types including adipose tissue, dental pulp, peripheral blood, placenta
and umbilical cord. These unique biological properties of MSCs highlight great
potential in several applications such as regenerative medicine, tissue engineering
and cell-based therapy (Wei et al., 2013).
Moreover, MSCs can secrete a number of bioactive molecules that affect to
biological changing of other cells or known as paracrine effect. These paracrine
Stem cells
effects are categorized into six main activities as immunomodulation, anti-apoptosis,
angiogenesis, supporting the growth and differentiation local stem and progenitor
cells, anti-scarring and chemoattraction (Meirelles Lda et al., 2009).
Several studies have shown that MSCs secreted a variety of angiogenic factors
including basic fibroblast growth factor (bFGF), vascular endothelial growth factor
(VEGF), placental growth factor (PIGF), monocyte chemoattractant protein 1 (MCP1) and interleukin 6 (IL-6). These paracrine factors are shown to promote local
angiogenesis that is important for tissue repair process. Additionally, MSCs secreted
large amounts of chemokines which play a role in recruitment of leukocytes to the site
of injury and further initiating the immune response. MSCs can limit the area of tissue
injury by their anti-apoptosis activity. VEGF, hepatocyte growth factor (HGF),
insulin-like growth factor 1 (IGF-1), Stanniocalcin-1, transforming growth factor beta
(TGF-β), bFGF and granulocyte-macrophage colony-stimulating growth factor (GMCSF) were found to reduce apoptosis of the normal tissues around the injured tissues.
Anti-scarring or anti-fibrotic is a one activity of paracrine factors secreted by MSCs.
Moreover, MSCs could support the growth of haematopoietic stem cells in vitro via
secretion of paracrine factors including stem cell factor (SCF), leukemia inhibitory
factor (LIF), IL-6 and macrophage colonystimulating factor (M-CSF) (Newman et al.,
2009).
Finally, MSCs possess immunomodulatory effects on both the innate and adaptive
immune systems by secreting a number of paracrine factors including secreted
prostaglandin E2 (PGE-2), TGF-, HGF, indoleamine 2,3-dioxygenase (IDO), LIF, MCSF, PGE-2, IL-6, IDO, TGF-β and PGE-2. These factors affect various biological
activities of the immune cells such as suppression of T cell proliferation, enhancement
of anti-inflammatory cytokines secretion, inhibition of dendritic cell maturation and
inhibition of NK cell proliferation. Taken together, all these activities are believed to
Stem cells
involve the therapeutic potency of MSCs that make them interesting for cell-based
therapy (Yi & Song, 2012).
8-Potential Use of Mesenchymal Stem Cells in Therapy:
The potential of MSC therapy involving their unique characteristics has
been
demonstrated
in
various
in
vivo
disease
models
and
has
shown
encouraging results for possible clinical use. In a clinical setting, MSCs are
now being explored in trials for various conditions, including orthopedic
injuries,
graft
transplantation
versus
(BMT),
host
disease
cardiovascular
(GVHD)
diseases,
following
autoimmune
bone
marrow
diseases,
and
liver diseases. Furthermore, genetic modification of MSCs to overexpress
antitumor genes has provided prospects for use as anticancer therapy in
clinical settings (Kim & Cho 2013).
 BMT and GVHD:
HSCT has been widely used over the past several decades to treat patients with
various malignant and nonmalignant diseases. However, the procedure remains
complicated
by
regimen-related
toxicity,
engraftment
failure,
and
GVHD.
Preconditioning regimens, such as chemotherapy and/or radiotherapy, may damage the
bone marrow and lead to a diminished engraftment of stem cells. MSCs are an
attractive therapeutic approach during or after transplantation as their transplantation
can minimize the toxicity of the conditioning regimens while inducing hematopoietic
engraftment and decrease the incidence and severity of GHVD (Tabbara et al., 2002).
GVHD is a severe inflammatory condition that results from immune-mediated
attack of recipient tissues by donor T cells during BMT. The clinical efficacy of MSCs
in acute GVHD (aGVHD) was first observed in a 9-year-old boy with steroid-resistant
Stem cells
grade IV aGVHD. The patient, who was unresponsive to other therapies, showed a
complete response after receiving haploidentical third-party MSCs (Le Blanc et al.,
2004). Following this pilot study, MSC treatment has been studied extensively in
steroid-refractory GVHD. Based on these properties, MSCs have been further
developed into an FDA-approved commercialized "off-the-shelf" product known as
Prochymal (Osiris Therapeutics Inc., Columbia, MD, USA), which is derived from the
bone marrow of healthy adult donors. Prochymal was used in a randomized
prospective study to treat patients directly after diagnosis of GVHD (Wu et al., 2011).
While studies on the use of MSCs for treatment of aGVHD have yielded promising
results, the therapeutic efficacy of MSCs in chronic GVHD (cGVHD) is less clear
because of the paucity of studies. While some studies indicated efficacy of MSCs,
even in cGVHD, others suggested that MSCs are less effective in cGVHD than
aGVHD. In studies of MSC therapy in both aGVHD and cGVHD patients, the
response rates were higher in aGVHD than cGVHD patients. In addition, the
infusion of MSCs following HSCT could prevent the development of aGVHD,
while the development cGVHD remained unaffected ( Kim & Cho, 2013).

Neurodegenerative diseases:
Amylotrophic lateral sclerosis: MSCs have the ability to differentiate into neurons
(Wang et al., 2010). The first MSCs transplantation for neurodegenerative disorder
was conducted in acid sphingomyelinase mouse model. After the injection of
MSCs, there was a decrease in disease abnormalities and improvement in the
overall survivability of the mouse. Based on this experiment, a study was designed
to ascertain the potency of MSC transplantation into amylotrophic lateral sclerosis
(ALS), a neurodegenerative disease that particularly degenerate the motor neurons
and disturb muscle functionality (Mazzini et al., 2003). The MSCs were isolated
Stem cells
from the bone marrow of patients and then injected into the spinal cord of the same
patients, followed by tracking of MSCs using MRI at 3 and 6 months. As a result,
neither structural changes in the spinal cord nor abnormal cells proliferation was
observed. However, the patients were suffering from mild adverse effects, i.e.
intercostal pain irradiation and leg sensory dysesthesia which were reversed in few
weeks duration. In another study, the AD-MSCs were genetically modified to
express GDNF and then transplanted in rat model of ALS which improved the
pathological phenotype and increased the number of neuromuscular connections
(Suzuki et al., 2008).
Parkinson's disease: Parkinson's disease (PD) is a neurodegenerative disorder,
characterized by substantial loss of dopaminergic neurons. The MSCs enhanced
tyrosine hydroxylase level after transplantation in PD mice model. MSCs by
secretion of trophic factors like vascular endothelial growth factor (VEGF), FGF-2,
EGF, neurotrophin-3 (NT3), HGF and BDNF contribute to neuroprotection without
differentiating into neurocytes (Wang et al., 2010). Other strategies are being
adopted like genetic modifications of hMSCs, which induce the secretions of
specific factors or increase the dopamine (DA) cell differentiation. BM-MSCs were
transduced with lentivirus carrying LMX1a gene and the resulted cells were similar
to mesodiencephalic neurons with high DA cell differentiation. Research group
from the university hospital of Tubingen in Germany first time delivered MSCs
through nose to treat neurodegenerative patients. The experiments were performed
on Parkinson diseased rat with nasal administration of BM-MSCs. After 4.5 months
of administration, MSCs were found in different brain regions like hippocampus,
cerebral, brain stem, olfactory lobe and cortex, suggesting that MSCs could survive
and proliferate in vivo successfully. Additionally, it was observed that this type of
administration increased the level of tyrosine hydroxylase and decreased the toxin
Stem cells
6-hydroxydopamine in the lesions of ipsilateral striatum and substantia nigra. This
delivery method of MSCs administration could change the face of MSCs
transplantation in future (Danielyan et al., 2014).
Alzheimer disease: Alzheimer disease (AD) is one of the most common
neurodegenerative diseases. Its common symptoms are dementia, memory loss and
intellectual disabilities. Till now no treatment has been established to stop or slow
down the progression of AD. Recently, researchers are in the search to reduce the
neuropathological deficits by using stem cell therapy in AD animal model. It was
demonstrated that human AD-MSCs modulate the inflammatory environment,
particularly by activating the alternate microglia which increases the expression of
Aβ-degradation enzymes and decreases the expression of pro-inflammatory
cytokines (Ma et al., 2013). Furthermore, it was observed that MSCs modulate the
inflammatory environment of AD and inadequacy of regulatory T-cells (Tregs) and
later on it was reported that they could modulate microglia activation. It was
previously demonstrated that human UCB-MSCs activate Tregs which in turn
regulated microglia activation and increased the neuronal survival in AD mice
model (Yang et al., 2013). Most recently, it was evidenced that MSCs enhanced the
cell autophagy pathway, causing to clear the amyloid plaque and increased the
neuronal survivability both in vitro and in vivo (Shin et al., 2014).

Autoimmune diseases:
MSCs are also used in immune disorders because MSCs have the capacity of
regulating immune responses. After revealing the facts that human BM-MSCs could
protect the haematopoietic precursor from inflammatory damage, other hMSCs can
be used for the treatment of autoimmune diseases (Riordan et al., 2007).
Stem cells
Rheumatoid arthritis: Rheumatoid arthritis (RA) is a joint inflammatory disease
which is caused due to loss of immunological self-tolerance. In preclinical studies
on animal models, MSCs were found helpful in the disease recovery and decreasing
the disease progression. The injections of human AD-MSCs into DBA/1 mice
model resulted in the elevation of inflammatory response in the animal. Following
the injections of AD-MSCs, the Th1/Th17 antigen-specific cells expansion took
place due to which the levels of inflammatory chemokines and cytokines reduced,
whereas this treatment increased the secretion of IL-10. Along with its antiinflammatory function, IL-10 is an important factor in the activation of Tregs that
controls self-reactive T-cells and motivates peripheral tolerance in vivo (Wehrens et
al., 2013). Similar to this, human BM-MSCs demonstrated the same results in the
collagen-induced arthritis model in DBA/1 mice. These studies suggest that MSCs
can improve the RA pathogenesis in DBA/1 mice model by activating Treg cells
and suppressing the production of inflammatory cytokines. However, some
contradictions were reported in adjuvant-induced and spontaneous arthritis model,
showing that MSCs were only effective if administered at the onset of disease. On
exposure to inflammatory microenvironment, MSCs lost their immunoregulatory
properties (Papadopoulou et al., 2012).
Type 1 diabetes: Type 1 diabetes is an autoimmune disease caused by the
destruction of β-cells due the production of auto antibody directed against these
cells. As a result, the quantity of insulin production reduces to a level which is not
sufficient to control the blood glucose. MSCs can differentiate into insulin
producing cells and have the capacity to regulate the immunomodulatory effects.
For the first time, nestin positive cells were isolated from rat pancreatic islets and
differentiated into pancreatic endocrine cells. Nestin positive cells were isolated
from human pancreas and transplanted to diabetic nonobese diabetic/severe
Stem cells
combined immunodeficiency (NOD-SCID) mice, which helped in the improvement
of hyperglycaemic condition (Zulewski et al., 2001). However, these studies were
found controversial and it was suggested that besides pancreatic tissues, other
tissues can be used as an alternative for MSCs isolation to treat type 1 diabetes.
Human BM-MSCs were found effective in differentiating into glucose competent
pancreatic endocrine cells in vitro as well as in vivo. It was demonstrated that UCBMSCs behave like human ESCs, following similar steps to form the differentiated
β-cells (Prabakar et al., 2012). Unsal et al.(2015) showed that MSCs when
transplanted together with islets cells into streptozotocin treated diabetic rat model
enhance the survival rate of engrafted islets and are found beneficial for treating
non-insulin-dependent patients in type 1 diabetes.

Cardiovascular diseases:
For myocardial repair, cardiac cells transplantation is a new strategy which is
now applied in animal models. MSCs are considered as good source for
cardiomyocytes differentiation. However, in vivo occurrence of cardiomyocytes
differentiation is very rare and in vitro differentiation is found effective only from
young cell sources (Ramkisoensing et al., 2011). MSCs trans-differentiated into
cardiomyocytes with cocktail of growth factors were used to treat myocardial
infarction and heart failure secondary to left ventricular injury. The systematic
injection of BM-MSCs into diseased rodent models partially recompensed the
infarcted myocardium (Nagaya et al., 2005). Roura et al. (2012) reported that
UCB-MSCs retained for several weeks in acute myocardial infarction mice,
proliferated early and then differentiated into endothelial lineage.
Stem cells

Liver diseases:
MSCs have been used to treat cirrhosis in a limited number of trials. Cirrhosis is
a chronic liver disease characterized by progressive hepatic fibrosis and loss of
hepatic structure with formation of regenerative nodules. Liver transplantation is
often the only option in advanced stage patients; however, it is limited by lack of
donors, surgical complications, and rejection. MSCs have the potential to be used
for the treatment of liver diseases due to their regenerative potential and
immunomodulatory properties (Ren et al., 2012). The MSC secretion profile also
represents an attractive property, as MSCs are known to secrete several anti-fibrotic
molecules such as hepatocyte growth factor (HGF) (Berardis et al., 2014).
Furthermore, MSC therapy could provide minimally invasive procedures with
relatively few complications, as compared to liver transplantation. In a phase I trial,
four patients suffering from end-stage liver cirrhosis were treated with autologous
MSCs and showed improved quality of life with no side effects during follow-up
(Mohamadnejad et al., 2007). In another phase I to II clinical trial, eight patients
with end-stage liver diseases received autologous MSCs. MSC administration was
well tolerated and improved liver functions. Thus, MSC therapy is safe, feasible,
and applicable in end-stage liver disease (Kharaziha et al., 2009).

Cancer:
MSCs are emerging as vehicles for cancer gene therapy due to their inherent
migratory abilities toward tumors. Whether MSCs themselves have antitumor
effects is still controversial as some studies have suggested that even unmodified
MSCs inhibit tumor growth and angiogenesis (Otsu et al., 2009), while others
report that MSCs promote tumorigenesis and metastasis (Karnoub et al., 2007).
Nonetheless, MSCs have been genetically modified to overexpress various
Stem cells
anticancer genes, such as ILs, IFNs, prodrugs, oncolytic viruses, antiangiogenic
agents, proapoptotic proteins, and growth factor antagonists, for targeted treatment
of different cancer types. The lack of safety mechanisms following MSC
administration has delayed the application of engineered MSCs in clinical settings.
Recently, a safety system to allow control of the growth and survival of MSCs has
been developed. The safety mechanism is a suicide system based on an inducible
caspase-9 protein that is activated using a specific chemical inducer of dimerization
(CID). Exposure to CID induced directed MSC killing within 24 hours. The
development of such safety mechanisms and their incorporation into MSC therapy
may allow extensive use of genetically engineered MSCs to treat cancer patients in
clinical settings ( Kim and Cho, 2013).

Bone fracture:
The osteogenic potential of MSCs has already been verified. MSCs have been
shown to readily differentiate down an osteogenic pathway in response to chemical
signals. MSCs have been shown to be the primary source for endochondral bone
formation, and as such are ideal for bone repair constructs. In a study of human
MSCs it was found that mechanical strain alone could induce osteogenic
differentiation (Sumanasinghe et al., 2006).
Two approaches have been used for cell delivery: bone marrow aspiration and
direct introduction at the lesion or expansion ex vivo before implantation.
Percutaneous autologous bone marrow grafting has been shown to be an effective
treatment for tibial diaphyseal nonunion in one study. The efficacy is influenced by
the amount of progenitor cells in the harvested graft (Hernigou et al., 2005).
Stem cells
The combination of mesenchymal stem cells, platelet rich plasma, and synthetic
bone substitute was found to be more effective in inducing new bone formation
(osteogenesis) than the use of platelet rich plasma combined with synthetic bone
substitute and the use of synthetic bone substitute alone (Kitaori et al., 2009).
Genetic manipulation of MSCs can be achieved by transduction using viral vectors
such as the adenovirus (Ad) or transfection by nonviral vectors such as liposomes.
Many investigators have tried to regenerate bone by transfecting MSCs with the
BMP gene. For example, Lieberman et al. (1999) have indicated that autologous
BMSCs expressing Ad-BMP2 can considerably promote segmental femoral defects
in rat models when compared with BMSCs expressing Ad-LacZ. It has been shown
that Ad-Runx2-MSCs transplanted in murine calvarial defects produce more bone
tissue compared to MSCs (Zhao et al., 2007). Recent studies have focused on
simultaneous application of BMPs and RUNX2. When these two factors were
entered into an immortal MSCs line and injected into mice, considerable bony
ossicle with marrow cavity was observed (compared to the application of cells that
expressed Ad-BMP2) (Yang et al., 2003).