SEMS Year 3 project Literature Review
Text 2
1. INTRODUCTION
1.1 Stem Cells
Stem cells are defined as single cells which have the capacity to undergo proliferation, self
maintenance of an undifferentiated phenotype and produce differentiated functional progeny
that give rise to specialised, mature cell types of embryonic and adult tissues. These cells
have biological properties to produce tissues and organs, with the ability to generate and
replace tissue cells through indefinite replication (Jukes et al., 2008, Potten and Loeffler et al.,
1990, Sell, 2004). Differentiation can be defined as the change in cellular phenotype which
depends upon its potency for example, totipotent, pluripotent, multipotent and unipotent. Self
renewal is regulated through the chromatin structure whereby polycomb group proteins
(PcG) repress transcription of gene that regulates differentiation (Jukes et al., 2008). Lastly,
proliferation is the division of cells leading to cyclic changes in gene expression (Potten and
Loeffler et al., 1990). Extensive researches in stem cells have been conducted as they could
be utilised as potential tools in medicine to cure diseased tissues and organs.
Stem cells can be divided into two main groups: embryonic and adult stem cells (ADSs).
Embryonic stem cells (ESCs) are derived from fertilised oocytes and are responsible for
embryonic and foetal development. When the fertilised oocytes undergo first division during
embryonic development they produce progeny that are considered as totipotent cells which
have the potential to form an entire organism (Thomson et al., 1998). The egg cell undergoes
symmetric division where the daughter cells receive the same chromosome as the original
and maintains cell phenotype. These daughter cells are known as blastomeres which form
clusters of cells with a hollow centre, known as blastocysts. Within the centre of the
blastocyst form a group of cells called the inner cell mass (ICM) that develops into the
embryo. During gastrulation process the blastocyst is organised to form the germinal cell
layers: ectoderm, mesoderm and endoderm. The totipotent ICM gives rise to pluripotent
ESCs and multipotent ADSs of the germ layers which produce progenitor cells that
differentiate into adult organs (Sell, 2004). These progenitor cells divide asymmetrically
producing one undifferentiated cell while the other daughter cell differentiates into a more
mature cell type.
ESCs are pluripotent cells capable of forming all three germ layers such as, pancreas, liver,
kidney, bone, epithelial, blood and many more. They undergo indefinite number of symmetric
divisions due to the presence of telomerase. The presence of the telomerase maintains
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telomere lengths at the end of chromosomes and extends replicative life span of stem cells
(Itskovitz- Eldor et al., 2000). Thomson et al., (1998) was the first study to derive human ES
cell lines from the ICM of blastocytes by the attachment to mouse embryonic fibroblast
feeder layer in vitro. They discovered that ESCs expressed cell surface markers such as,
stage specific embryonic antigen (SSEA-4) and alkaline phosphatase for undifferentiated
cells. Other supporting studies have found that transcription factor Oct-4 was an essential
characteristic for pluripotent cell lineages (Reubinoff et al., 2000). Reubinoff et al., (2000)
was the first to publish somatic differentiation of ESCs under prolonged cultivation. They
observed the formation of aggregates and the clusters of cells contained differentiated cells
expressing neural adhesion molecules (N-CAM). Both studies found that the ESCs did not
grow or differentiate when leukemia inhibitor factor (LIF) was present within the culture layer.
However, the use of human ESCs are ethically controversial. The problem associated with
the transplantation of embryonic stem cells into the body is formation of cancerous tumors
due to its unlimited proliferation potential. Thomson et al., (1998) observed teratomas
formation when human ES lines that were cultured in vitro for 4 to 5 months were injected
into
mice.
This
was
a
common finding between studies culturing ES cell lines and the teratomas that formed
contained derivatives of all three embryonic germ layers (Reubinoff et al., 2000).
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Figure 1.1. Different types of proposed stem cell niches (Jukes et al., 2008)
ADSs are derived from adult organs which are considered multipotent due to its ability to
form multiple cell types such as, bone, cartilage, marrow stroma, muscles and ligaments.
Source of these cells can be found most commonly in the bone marrow, peripheral blood and
umbilical cord blood (Kaufman et al., 2001). A well characterised stem cell population within
the adult stem cell is the hematopoietic stem cell population which give rise to all blood cell
lineages (Weissman et al., 2000, Kaufman et al., 2001). Baum et al., (1992) identified the cell
surface marker Thy-1 CD34 which contained pluripotent HSC progenitors, commonly used
for isolation.
ASCs reside in niches which are specific locations of tissues that enable cells to proliferate
and produce differentiated progeny but also remain their quiescent state. However, when
stem cells migrate away from its niche, it differentiates into progenitor or precursor cells and
no longer maintains its undifferentiated phenotype (Jukes et al., 2008, Weissman and
Wagers, 2004). There are complex interactions between the stem cells and the
microenvironment involving transduction of intercellular signals. It has been reported that
bone morphorgenic protein (BMP) receptors directly control differentiation in germ-line stem
cells (Ohlstein et al., 2004). Maintaining quiescence of HSCs involves intrinsic and extrinsic
cues, such as, Notch ligands and Cdc42 (Yang et al., 2007) as well as spindle shaped NCadherin CD45- osteoblastic (SNO) cells found on the surface of bones have been reported
to support long term survival of HSCs within the stem cell niche (Zhang et al., 2003).
ASCs in particular are therapeutically beneficial cells which can be used in autologous tissue
transplantation such as bone marrow transplantation. The bone marrow also contains a
subset of non-hematopoietic stem cells, known as mesenchymal stem cells (MSCs) (Salem
and Thiemermann, 2010). The research on MSCs provides an area of interest in the
regeneration of the muscular skeletal tissue in vivo due to its multilineage potential.
1.2 Mesenchymal Stem Cells
1.2.1 Origin of MSCs
Adult bone marrow contains a heterogeneous population of cells including HSCs,
macrophages, adipocytes, epithelial cells and stromal cells (Salem and Thiemermann, 2010).
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The stromal population exert distinctly different functions to the HSCs and produce nonhematopoietic cells including fibroblasts, adipocytes, ostoblasts and chondrocytes (Pittenger
et al., 1999). The first identification of subpopulations within the stromal cell was
acknowledged by Friedenstein (1970) who cultured bone marrow explants and found a small
population of cells in vitro that developed into adherent colony forming units and observed
cells resembling fibroblastic morphology after 9-12 days in culture. These cells were
described as colony forming unit- fibroblasts (CFU-F) (Freidenstein et al., 1976) and it was
suggested that fibroblast colonies arised from the proliferation of a single CFU-F (CastroMalaspina et al., 1980). It was found that the adherent cells could also be used as a feeder
layer for HSC growth, controlling differentiation and proliferation during hematopoiesis
(Caplan et al., 1991, Dexter et al., 1977) and exhibited regeneration properties to bone tissue
in transplants (Freidenstein et al., 1976). These cells were termed as ‘Mesenchymal stem
cells’ in which they originate from the mesodermal germ layer and have potential to
differentiate into lineages of the mesenchymal tissue and the ability to proliferate as
undifferentiated state (Kuznetsov et al., 1997).
1.2.2 Isolation and expansion of MSCs
There are many reported sites in which the MSCs can be isolated and cultured from for
example, bone marrow, placenta, fat, umbilical cord, brain and muscles (Bianco et al., 2001,
Jiang et al., 2002). Freidenstein et al., (1970) isolated bone marrow stromal stem cells
(BMSSCs) by relying on the cells’ ability to rapidly adhere to the surface of the culture flask
which is a distinct property of MSCs. Other studies have also used density cut suspension
technique to separate hematopoietic cells from stromal cells by isolating cells below a
density of 1.070g/ml (Castro-Malaspina et al., 1980). The isolated cells grew as fibroblastic
cells which developed colonies and only the cells that were attached were obtained
(Pittenger et al., 1999). However, the stromal cells gave a variation in cell density and
contained cell surface markers that did not represent MSC characteristics suggesting a
heterogeneous population of cells (Castro-Malaspina et al., 1980, Halleux et al., 2001). MSC
population is rare in the bone marrow, representing approximately 0.001% of the nucleated
cells (Chamberlain et al., 2007).
For a homogeneous MSC population, studies have used monoclonal antibody STRO-1 to
isolate sub-populations of progenitor cells containing CFU-F (Gronthos et al., 2003) and
vascular cell adhesion molecule- 1 (VCAM-1) or CD106 as positive markers for cell-matrix
interactions in vivo (Kolf et al., 2007). CD105 have been recognised as an antigen which is
positively expressed by the MSC population which separates it from the hematopoietic cell
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population (Majumdar et al. 2000). Flow cytometry have been equipped to yield different
subsets of MSC population. D’Ippolito et al. (2006) discovered marrow-isolated adult
multilineage inducible (MIAMI) cells through clonal expansion under 3% oxygen. These cells
expressed pluripotency by the ability to differentiate along the mesodermal and endodermal
lineages. Similarly, multipotent adult progenitor cells (MAPC) display pluripotent features as
they were found to undergo extensive population doubling and have a broader differentiation
potential than MSCs. These cells were isolated and purified using negative markers for flow
cytometry (Reyes et al., 2001, Zimmermann et al., 2003). Other populations include recycling
stem cells (RS-1 and RS-2) which are cells that proliferate rapidly and produce mature
mesenchymal cells (Colter et al., 2000).
1.2.3 Differentiation of MSCs
Mesenchymal stem cells are considered multipotent with a capacity to differentiate along a
range of mesenchymal cell-lineages, for example, chondrocytes, osteoblasts, myoblasts and
adipocytes (Chamberlain et al., 2007). Osteogenic differentiation of MSCs in vitro was
conducted on mononuclear fraction of human MSCs with osteogenic medium containing
dexamethosome and found that there was an increase in alkaline phosphatase activity and
deposition of calcified extracellular matrix (ECM) containing hydroxyapatite during the 16
days of culture (Jaiswal et al., 1997). Also, the presence of progenitor cells that could
differentiate into osteogenic cells were supported by positive expressions of osteocalcin
(Sudo et al., 2007). For chondrogenesis, transcription factors such as SOX-9 and ECM gene
for collagen type I and IX have been positively characterised in MSC-derived chondrocytes
(Kolf et al., 2007, Lefebvre and Crombrugghe, 1998,). Matsuda et al., (2005) induced
chondrogenic differentiation to MSCs by pellet cultivation with bone morphorgenic proteins
(BMPs) and transforming growth factor -β (TGF-β) which resulted in an increase in the
degree of expression of aggrecan mRNA. Furthermore, the MSCs can also undergo
adipogenic differentiation. Clonal analysis of human MSCs showed that the colonies were
positive for Oil Red-O staining and there was an increase in the number of lipid vacuoles per
colonies (Sekiya et al., 2004). An indicator of adipogenesis is perioxisome proliferatoractivated receptor γ2 (PPAR-γ2) which reverses osteoblastic phenotype and lipoprotein lipase
(LPL) is expressed as a late marker of adipogenic differentiation (Pittenger et al., 1999, Sudo
et al., 2007).
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MSCs, similar to somatic cells, have limited lifespan when grown in vitro unlike ESCs. MSCs
undergo a certain number of divisions, 230-250 population doublings, before it enters
senescence phase where the cells lose its proliferative capacity and becomes inactive. This
is known as the ‘Hayflick’s limit’, first proposed by Hayflick in 1965. The benefit of this is to
prevent the transmission of accumulated mutations to the daughter cells during replication.
(Colavitti and Finkle, 2005). There is a strong association between cell senescence and the
oxygen tensions within a stem cell environment which may play an important role in the
expansion of cells. This will lead to further research on the main principles of metabolic
system in order to see how oxygen is utilised by the cells.
1.3 Energy Metabolism
1.3.1 Energy Metabolism in cells
Energy metabolism involves complex chemical changes which occur in the cells or body of
an organism. The primary objective of metabolism is to maintain a steady supply of energy
source so that the living cells can reproduce and grow (Salway, 1994). Energy is required for
both metabolic reactions and physiological functions and it involves catabolic and anabolic
reactions where oxidation and reduction of organic molecules are maintained together
(Mattson, 2003). The main substrate that is metabolised is glucose and other substrates
include; carbohydrates, lipids and proteins and the energy which is released from the
oxidation of substrates is converted into small, water-soluble molecules of adenosine
triphosphate (ATP). ATP is a temporary energy source and when it is hydrolysed, it
generates a large amount of free energy which is directly used in intracellular energy yielding
and requiring reactions (Rastogi, 2003). The sites of action for energy metabolism are cell
cytoplasm and the mitochondria.
Initial substrates are broken down into their constituent components in order to be utilised for
the production of ATP. Proteins are broken down into amino acids; alanine, cysteine and
glutamine, which are converted into pyruvate and oxaloacetate which enter the Krebs’ Cycle.
Lipids are separated into fatty acids and glycerol, where the fatty acetyl CoA synthase
converts fatty acids to fatty acetyl CoA via the beta oxidation process. Once the fatty acetyl
CoA is transported into the cytosol of the mitochondria, it is broken down into acetyl CoA and
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smaller bi-products. The acetyl CoA and reduced nicotinamide adenine dinucleotide (NADH)
are utilised and the energy is used in pathways to generate ATP (Brownie, 2005).
There are two ways in which ATP is synthesised within the cells. Firstly, metabolic processes
release energy to be used to synthesise ATP from phosphorylation of adenosine
diphosphate (ADP) and inorganic phosphate (Pi). This process occurs under the absence of
oxygen, known as substrate-level phosphorylation. (Salway, 1994). Secondly, the oxidisation
of reduced NADH and FADH2 in the presence of oxygen is known as oxidative
phosphorylation (Rastogi, 2003).
1.3.2 Glycolysis
Oxidation of glucose takes place during glycolysis and this process occurs in the cytoplasm
of the cell. Glucose is phosphorylated, making the glucose more reactive therefore enabling
it to be broken down into two molecules of Triose Phosphate (TP). For every one molecule of
glucose, two ATP and two molecules of pyruvate are produced. In this process, the hydrogen
molecule is removed from the TP and transferred to two hydrogen carrier molecule
nicotinamide adenine dinucleotide (NAD) where they become reduced to NADH. Pyruvate
contains a large chemical potential energy and with free oxygen being available, some of the
energy can be released via the Kreb cycle and oxidative phosphorylation. However,
glycolysis can occur in the absence of oxygen and when this occurs, NADH converts back to
NAD which is reused in glycolysis and pyruvate is converted into lactate. The metabolism in
the absence of oxygen is also known as substrate phosphorylation. (Jones et al. 2001)
1.3.3 Krebs’ Cycle
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Figure 1.2. Schematic diagram of the link reaction and the Krebs’ cycle (Jukes et al.,
2008)
Krebs cycle is a closed pathway which is controlled by enzymic reactions. The two pyruvates
that are generated during glycolysis pathway is converted into acetyl CoA and oxaloacetate,
once it diffuses from the cytoplasm into the mitochondria. (Salway, 1994). Oxidation of
pyruvate releases free energy which is initiated by the enzymes present in the mitochondria.
The acetyl CoA and the oxaloacetate condense to form citrate which enters the Krebs cycle
and this involves a series of decarboxylation and dehydrogenation reactions. Hydrogen is
removed and is passed to the coenzymes such as NAD and flavin adenine dinucleotide
(FAD). This produces reduced NADH and FADH2 which carry the hydrogen through to the
Electron transport chain (Salway, 1994). This is considered as the most important
contribution made by the Krebs cycle as the released hydrogens are necessary for the
production of ATP. For each cycle, two carbon dioxide molecules, one reduced FADH2 and
three reduced NADH and one ATP molecules are yielded. The Krebs cycle does not utilise
molecular oxygen but it is necessary during oxidative phosphorylation (Jones et al., 2001).
1.3.4 Electron Transport chain
The reactions that occur in the ETC generates majority of the energy via the phosphorylation
of ADP to ATP and the site of reaction is the mitochondrial membranes. NADH and FADH2
are reoxidised by the enzymes located on the cristae of the inner mitochondrial membrane.
The hydrogen atom splits into a hydrogen ion (H+) and an electron, in which the electron
passes through a series of electron carriers generating a proton gradient. Electrons from
either complex I or II,
NADH-ubiquinone oxidoreductase and succinate-Q oxidoreductase
respectively are passed via complex III, ubiquinone-cytochrome c oxidoreductase,
to
complex IV, cytochrome oxidase, which then drives ATP synthesis by ATP synthase at
complex V (Lambert and Brand, 2007). The transportation of electron through the carriers
releases energy that is used to phosphorylate ADP into ATP.
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1.3.5 Warburg Effect and Pasteur Effect
Cells usually undergo oxidative phosphorylation to generate ATP in aerobic conditions.
Alternatively glycolysis pathway is utilised when the oxygen levels are low and this is known
as Pasteur effect. Wang et al., (2005) found that the production of lactate in hADSCs was
increased under hypoxia suggesting a shift towards glycolysis. Human blastocytes also have
high glycolytic capacity due to the low oxygen nature in situ but 75-85% of the ATP is
generated via oxidative phosphorylation (Power et al., 2008). A change in energy metabolic
pathway was observed in MEFs upon transferral from a 3% to 20% oxygen tension.
Chondrocytes generate energy via glycolysis, known as Warburg effect where the cells
utilise glycolysis despite being placed under high oxygen environments (Heywood and Lee et
al., 2008). ESCs are also able to utilise glycolysis under aerobic conditions. However, once it
differentiates, different lineages are expected to experience a change in energy metabolism
(Power et al., 2008). Importantly, Grayson et al.,(2006) found that hMSCs underwent
glycolysis in high oxygen tensions. Under hypoxic conditions, the ratio of glucose
consumption to lactate production increased which showed that glycolytic pathway was
favoured.
1.4 Oxygen and Stem Cells
1.4.1 Oxygen tension in vivo tissue
The oxygen tension within the biological niche of stem cells varies between the tissues.
Human mesenchymal stem cells reside in the mononucleated fraction of the bone marrow
and the oxygen pressure in the compartment ranges between 35-49mmHg, corresponding to
a tension of 4-7% (Grant and Smith, 1963, Lennon et al., 2001). Hence, these cells reside
under low oxygen tensions, known as hypoxic conditions. Cells surrounding the sinus
compete for oxygen and nutrients in the marrow; hence oxygen is depleted further away from
the sinus. It is found that the in vivo oxygen tension for adipose- derived MSCs extracted
from the stromal vascular fraction of the lipo-aspirated tissue is approximately 3%, despite
high vascularity (Ma et al., 2009). Stem cells are able to maintain their quiescent state and
plasticity in hypoxic conditions (Holzwarth et al., 2010). Exposure to higher oxygen
concentrations cause the cells to differentiate to a more mature state which are better
adapted and resistant to higher oxygen levels (Ivanovic, 2009). Chondrocytes also reside in
low tensions in situ, where the oxygen levels are as low as 1% (d’Ippolito et al.,2006,
Heywood and Lee et al., 2008). Stem cells also utilise oxygen as a signalling molecule to
influence stem cell survival, proliferation and differentiation.
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1.4.2 Telomere and Telomerase
The expansion of cells through proliferation in vitro have become a vital step as the demands
for
expanded cells for repair-regeneration of tissues are greatly enhanced over time.
However, the principle of indefinite replication is limited by the lifespan of the cell which is to
some extent dependent on the structures that are present at the ends of the chromosomes,
known as telomeres (Colavitti and Finkel, 2005). Telomeres consists of repetitive non-coding
DNA sequences, TTAGGG, in which the number of repeats on any chromosome may vary
and these sub-units form a loop structure which cap the ends of the chromosome (Graakjaer
et al., 2007). This mechanism prevents the cells from being exposed to degradation
processes, for example, chromosomal fusion, recombination and terminal DNA degradation
(Hiyama et al., 2004). However, when cells undergo successive population doubling via DNA
synthesis and cell division, the telomere sub-units shorten respectively. The gradual
shortening of the telomere length continues until it reaches a critical length in which the cells
begin to undergo cell cycle arrest and triggers a p53 mediated senescence pathway
(Graakjaer et al., 2007). The result of telomere shortening acts as a marker for cellular
ageing (Yanada et al., 2006) and loss of cell viability (Wright and Shay, 2005). The main
gene marker for senescence is p53 and it is suggested that telomere shortening could initiate
the p53-dependent pathway resulting cells to senesce (Zglinicki et al., 1995). Hence, the
telomere attrition causing cell senescence due to lack of telomerase activity is known as
‘replicative senescence’ (Moussavi-Harami et al., 2004).
Certain cells do not have limited proliferative capacity even when the cells undergo
population doubling. In particular immortalised cells, for example, ESCs and cancerous cells
are capable of maintaining their telomere length and function throughout their lifetime
through the activity of the enzyme, telomerase which is activated by the reverse transcriptase
protein encoded by the TERT gene (Moussavi-Harami et al., 2004). Telomerase can add
telomere repeats onto the chromosome ends which enhances self-renewal capacity and cells
can undergo large numbers of proliferation without inducing senescence (Hiyama et al.,
2004). Harley et al. (1990) found that when non-transformed human fibroblasts were
continuously passaged, the length of telomeric DNA decreased and found that the loss of
length was dependent on the number of divisions the cells had undergone. Most importantly,
Simonsen et al. (2002) did not detect the presence of telomerase in hMSCs and found that it
was subjected to continuous telomere attrition. In comparison to hMSCs, murine MSCs
contained telomerase which enabled the cells to undergo more than 100 population
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doublings (Hiyama et al., 2004). Whereas, in certain sub-populations of primitive MSCs,
known as MAPCs, telomere attrition is preserved during cell expansion which suggests that
these cells may contain the telomerase enzyme (Zimmermann et al., 2003). The introduction
of hTERT expression into MSCs has been carried out by researchers. Telomere positive
hMSC-telo1 cells confirmed an overall increase in telomere length and also extended the
lifespan without changing phenotypic characteristics (Graakjaer et al., 2007).
1.4.3. Cell Senescence and Reactive Oxygen Species
Cellular senescence is a complex biological change in which the cell loses its proliferation
capacity after a certain number of population doublings despite being viable and
metabolically active (Afshari et al., 1994, Chen et al., 1995). Whilst there are evidence that
telomere shortening triggers DNA damage that mediates cell cycle arrest or early
senescence, telomere- independent mechanisms that limit proliferative lifespan also exists,
known as premature senescence. Premature senescence are triggered by extrinsic factors
that limit the replicative lifespan of a cell by inducing a senescent phenotype (Simonsen et al.,
2002, Mathon and Lloyd, 2001). Reactive oxygen species (ROS) are a variety of reactive
molecules and free radicals which are formed in the mitochondria as a by-product of
oxidative phosphorylation (Busuttil et al., 2004). The electron leak within the ECT causes
partial reduction of oxygen which generates superoxide anion molecules. Superoxide anion
is a precursor of most endogenous ROS which induces intracellular oxidative damage to
DNA, proteins and lipids (Chen et al., 2008). The production of ROS is inevitable as it is a
part of the essential aerobic metabolism that occurs in mammals. To overcome the
accumulation of ROS, the organism possesses a defence mechanism including an array of
enzymatic and non-enzymatic antioxidants to protect the cells, for example, catalase,
superoxide dismutase (SOD), ascorbate and gluthathione perioxidase (Lennon et al.,2001,
Turren et al., 2003). Superoxide anion is converted into hydrogen peroxide by SOD followed
by further decomposition into water and oxygen by catalyse and glutathione peroxidise
(Chen et al., 2008, Turren et al., 2003). However, when the balance between aerobic
metabolism and antioxidant defence system disrupts, the accumulation of ROS causes the
cells to experience oxidative stress (Chen et al., 1995). Consequences of high oxidative
stress levels can be detrimental to the cells as it causes mutations in the DNA leading to
cellular degeneration and ultimately cell death. Also, it can induce proliferative arrest as it
accelerates the loss of telomere length and induces early premature senescence (Busuttil et
al., 2003, Zglinicki et al., 1995). Senescent cells are identified by the change in morphology
and by beta-galactosidase activity (Colavitti and Finkel, 2005). Under 3% oxygen, hMSCs
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maintained spindle shape morphology, however, under long term culture, the cells adapted
an enlarged flattened phenotypic appearance which suggests that the cells have undergone
senescence. Elevated numbers of beta-galactosidase positive cells was observed in 20%
oxygen compared to lower oxygen environments (Fehrer et al.,2007).
1.4.4. Oxygen tension during proliferation
The significance of low physiological oxygen tensions in vivo help regulate cell survival and
determine the energy metabolism of the cells. Various studies have found that hypoxia
extends the replicative lifespan of cells in culture. Parrinello et al., (2003), found that cultured
MEFs in 3% oxygen increased proliferation up to 60PD and in other studies, the stem cell
transcription factor, NANOG, which is responsible for self renewal was decreased under
normoxic conditions (Fehrer et al., 2007). Larger CFU-F numbers in primary cultures were
observed at low oxygen tensions in primary murine MSCs cultured at 5% oxygen (Grayson et
al., 2006). The prolonged lifespan of cells and proliferation rate is also directly linked to ROS
generation within the cells. Packer and Fuehr (1977) found that hypoxia decreased the
production of ROS in human fibroblasts resulting in low levels of oxidative damage to the
DNA. Furthermore, Lennon et al.,(2001) found that DNA numbers were much lower in
cultures that were primarily expanded under normoxic conditions and even for cells that were
cultured under normoxia after expansion under low oxygen conditions. In low oxygen
tensions of hMSCs, remodelling capabilities were enhanced with an increase in production of
VEGF, MMP-3, GAG synthesis and with the up regulation of fibronectin only observed in
hypoxic conditions (Grayson et al., 2006). However, a study on WI-38 fibroblasts showed
that under normoxic conditions proliferation ceased after 44-45PD whereas proliferation was
inhibited by hypoxia (Zglinicki et al.,1995). In terms of energy metabolism for MSCs, the
utilisation of glycolysis was increased under low oxygen conditions (Grayson et al., 2006,
Wang et al., 2005). Low oxygen cultures are also associated with the induction of hypoxic
inducible factor (HIF-1α). Hypoxia is found to utilise the mitochondrial ROS as a signalling
molecule to activate the HIF-1α which induces telomerase activity and telomerase reverse
transcriptase mRNA. Therefore, high ROS generation stimulates the activation of the
transcription factor in order to prevent replicative senescence (Bell et al., 2007).
1.4.5. Oxygen tension during differentiation
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Several studies have assessed the effect of hypoxia on differentiation of
stem cells. Differentiation of rat derived MSCs (rMSCs) into the osteogenic lineage
was enhanced under
5% oxygen culture in osteogenic media through calcium content measurement.
The levels of alkaline
phosphatase
(AP)
also
increased
when
oxygen
tensions were switched from normoxic to hypoxic conditions (Lennon et al.,
2001). In normoxic conditions, a peak rise in AP was observed whereas under
hypoxia, the levels increased continuously (Grayson et al.,2006). There are
studies that disagree on the relationship between osteogenesis and hypoxia.
D’Ippolito et al.,(2006) studied MIAMI cells grown in hypoxic conditions and
found that certain prototypic markers of osteoblasts, for example, RunX2,
BSP
and
OCN expressions were inhibited, despite being
cultured in
osteogenic medium. Chondrogenic differentiation of ovine MSCs in 5% oxygen
produced higher collagen type 2 expression and had 1.4 fold higher sulphated
glycosaminoglycan concentration when compared to cultures at 20% oxygen
(Krinner et al., 2009). A study conducted by Wang et al., (2005) found that the
rate of glycolysis increased after chondrogenic differentiation of hADSCs.
Whereas, an increase in oxygen consumption was reported for bone marrow
derived MSCs during adipogenic differentiation (Chen et al., 2008). The effect
of adipogenic differentiation in adipose tissue derived MSCs under hypoxia
was inhibited but exposure to normoxic conditions afterwards enhanced the
differentiation (Valorani et al., 2010).