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209
research article

The concept of radiation-enhanced stem cell

differentiation
Adam A. Mieloch and Wiktoria M. Suchorska

Radiobiology Laboratory, Department of Medical Physics, The Greater Poland Cancer Centre

Radiol Oncol 2015; 49(3): 209-216.

Received 9 March 2015

Accepted 5 June 2015
Correspondence to: Dr. Wiktoria M. Suchorska, Radiobiology Laboratory, Department of Medical Physics, The Greater Poland Cancer Centre,
15th Garbary Street, 61-866 Poznan, Poland. Phone: +48 618 850 477; [email protected]
Disclosure: No potential conflicts of interest were disclosed.

Background. Efficient stem cell differentiation is considered to be the holy grail of regenerative medicine. Pursuing
the most productive method of directed differentiation has been the subject of numerous studies, resulting in the de-
velopment of many effective protocols. However, the necessity for further improvement in differentiation efficiency
remains. This review contains a description of molecular processes underlying the response of stem cells to ionizing
radiation, indicating its potential application in differentiation procedures. In the first part, the radiation-induced dam-
age response in various types of stem cells is described. Second, the role of the p53 protein in embryonic and adult
stem cells is highlighted. Last, the hypothesis on the mitochondrial involvement in stem cell development including its
response to ionizing radiation is presented.
Conclusions. In summary, despite the many threats of ionizing radiation concerning genomic instability, subjecting
cells to the appropriate dosage of ionizing radiation may become a useful method for enhancing directed differen-
tiation in certain stem cell types.

Key words: stem cells; ionizing radiation; differentiation; regenerative medicine; tissue engineering

Introduction division, which simultaneously increases the num-

ber of cells in the niche and the number differenti-
Stem cells (SCs) possess a set of unique advantag- ating into tissue specific lineages, providing cells
es, including the ability to replicate and the ability required for tissue regeneration.3,4
to differentiate into many different types of cells, Stem cells have had a significant impact on the
called pluripotency. Due to the pluripotent char- progress of many fields of biotechnology, includ-
acteristic of these cells, they play a pivotal role in ing cell-based regenerative therapies, drug testing
tissue development and maintenance by replen- and screening, disease modeling, side effects in
ishing the depletion of cells caused by damag- radiotherapy and many more. In 2006, Yamanaka
ing factors or that occurs physiologically during et al. announced a breakthrough finding in regen-
tissue turn-over.1 The majority of recent studies erative medicine, describing the reprogramming
have mainly focused on two types of stem cells: of mouse adult fibroblasts into induced pluripo-
Embryonic stem cells (ESCs) and adult stem cells tent stem cells (iPSs) by introducing four factors:
(ASCs), also known as somatic tissue or mesenchy- Oct3/4, Sox2, c-Myc, and Klf4. iPSs in many aspects
mal stem cells.2 ESCs are derived from the inner resemble ESCs.5 This discovery solved many of the
cell mass of the blastocyst and are capable of dif- ethical disputes concerning the procurement of
ferentiating into the three embryonic germ layers: ESCs from human embryos and began a new era in
ectoderm, mesoderm and endoderm, thus contrib- regenerative medicine.
uting to the formation of almost every cell type. Since then, many attempts to harness the pluri-
ASCs reside in tissue-specific niches in a quiescent potency of stem cells into directed differentiation
state. Upon activation, they undergo asymmetric have been successful.6,7 Some of developed pro-

Radiol Oncol 2015; 49(3): 209-216. doi:10.1515/raon-2015-0022

210 Mieloch AA and Suchorska WM / Radiation-enhanced stem cell differentiation

tocols require the formation of embryoid bodies functional mechanism of DNA damage repair is
(EBs) prior to further differentiation. EBs are three- crucial for the maintenance of genomic stability.
dimensional cellular aggregates obtained by spon- The most dangerous type of DNA lesions are
taneous differentiation of ESCs or IPSs. EBs con- double strand breaks (DSBs), which are usually
sist of ESCs that are mostly differentiated into the caused by IR or free radical exposure. Repair of
embryonic germ layers: ectoderm, endoderm and DSBs is driven by two major pathways: homolo-
mesoderm.8 The differentiation process in many gous recombination (HR) and non-homologous
aspects mimics early mammalian embryogenesis, end joining (NHEJ).19 In the process of HR, sister
including cell to cell interactions. Moreover, the chromatids serve as a template; thus, the repair is
most essential method of EBs formation is based on considered error-free. NHEJ does not utilize sister
suspension culture deprived of antidifferentiation chromatids as a template and is therefore signifi-
factors. Due to simple methodology and similarity cantly more prone to error introduction. Depending
to embryogenesis, EBs are widely utilized as an in- on the phase of the cell cycle, one of the pathways
termediate stage during in vitro differentiation of is used predominantly. The requirement for sister
both human and murine ESCs. 9 chromatids in HR restrains its activity to the S and
Currently, many stem cell-based therapies are un- G2 phases. The NHEJ response dominates through
dergoing clinical trials, for example, Intravenous the rest of the cell cycle.20 There are also other types
Stem Cells After Ischemic Stroke, Human of DNA damage repair mechanisms: nucleotide ex-
Neural Stem Cell Transplantation in Amyotrophic cision repair (NER), base excision repair (BER) and
Lateral Sclerosis (ALS) and Treatment of Knee mismatch repair (MMR). However, their contribu-
Osteoarthritis by Intra-articular Injection of Bone tion to radiation-enhanced differentiation seems
Marrow Mesenchymal Stem Cells.1012 These trials to be negligible and will not be considered in this
are just a few from a still enlarging group of stud- study.
ies investigating the potential applications of stem
DNA damage repair in embryonic stem cells
cells in regenerative medicine, which indicates a
(ESCs)
growing need for reliable methods of directed dif-
ferentiation of SCs. It has been proven that the mechanisms of DNA
Ionizing radiation (IR) has been used for many damage repair in ESCs are more efficient compared
years as a basic tool in cancer treatment.13 The re- to other cell types.21 ESCs display a unique cell cy-
sponse of non-stem cells to irradiation has been cle structure. The G1 phase is significantly short-
extensively investigated by a number of studies, ened and the G1 to S transition is facilitated in or-
and to date, many molecular mechanisms of this der to promote rapid self-renewal. Consequently,
phenomena have been thoroughly elucidated.1416 the majority of the ESC population is in the phases
However, based on the current understanding con- of cell cycle where sister chromatids are available
cerning non-SCs, the radio-response of SCs cannot for use as a template. Due to this phenomena, ESCs
be anticipated and it could result in unexpected predominantly utilize high-fidelity HR.22
outcomes. ESCs serve as a pool of cells for the development
Radiation-induced differentiation has already of the whole organism. Therefore, DNA repair in
been reported in multiple studies17,18; however, it these cells requires high efficiency and accuracy in
has not been investigated as a potential tool in stem order to provide genomic stability. In the case of in-
cell differentiation protocols. The main goal of this sults in the genomic DNA that cannot be repaired,
review is to present research based indications that the cell undergoes apoptosis, which is significantly
radiation-enhanced differentiation is a promising facilitated by a mechanism known as mitochon-
technology for further development of stem cell drial priming in ESCs.23 Mitochondrial priming is
engineering. determined by the equilibrium between levels of
anti-apoptotic and pro-apoptotic proteins of the
B-cell lymphoma 2 (Bcl-2) protein family. ESCs
Radiation-induced DNA damage response
display elevated levels of pro-apoptotic proteins
in stem cells
within the mitochondria. Consequently, the initia-
Radiation-induced damage to genomic DNA trig- tion of apoptosis requires a considerably weaker
gers a cascade of biochemical reactions known as stimuli in order to cross the apoptotic threshold.
the DNA damage response (DDR), which includes This phenomenon ensures elimination of geneti-
cell cycle arrest, DNA repair and, in the case of un- cally unstable cells and prevents further transmis-
manageable lesions, senescence or apoptosis. The sion of mutations.

Radiol Oncol 2015; 49(3): 209-216.

 Mieloch AA and Suchorska WM / Radiation-enhanced stem cell differentiation 211

A previous study by Sokolov and Naumann re- actions and associations with various molecular
vealed that human embryonic stem cells (hESCs) processes has left many novel functions of this pro-
undergo apoptosis after relatively low-dose irra- tein remaining to be discovered.
diation. In the study, a 1.0 Gy dose of X-ray radia- p53 is a tumor suppressor protein responsible
tion triggered robust apoptosis. Conversely, doses for the induction of reversible cell cycle arrest,
of 0.5 Gy and 0.2 Gy did not increase the apoptotic which enables DNA repairs to be conducted, and
response.24 A study by Lan et al. reported that a 2.0 the initiation of apoptosis in the case of irreversible
Gy dose of X-ray radiation resulted in an almost DNA damage. p53 is a transcription factor that,
60% decrease in the survival rate of hESCs 5 days upon activation, binds to the promoters of target
post-irradiation. The same study found that X-ray genes, either inducing or repressing their tran-
irradiation elevated metabolic activity (XTT assay) scription depending on the gene.33 p53 can trigger
1.5-fold after a 2.0 Gy dose and 2.5-fold after a 5.0 apoptosis via two pathways: the transcriptional
Gy dose. The same dosage of 2.0 Gy and 5.0 Gy (intrinsic) pathway, as described above, or the non-
resulted in elevated levels of reactive oxygen spe- transcriptional (mitochondrial) pathway by direct
cies (ROS) and nitrogen (RNS) species for 1 week interactions with pro- and anti-apoptotic proteins.
following exposure.25 The main target genes for its proapoptotic activ-
ity include p53 upregulated modulator of apopto-
DNA damage repair in adult stem cells (ASCs)
sis (Puma) and Bcl-2-associated X (Bax) proteins,
ASC sensitivity to irradiation varies greatly, de- which belong to the Bcl-2 family.34 DNA damage
pending on their type and developmental stage. results in ataxia telangiectasia mutated (ATM) pro-
However, it is postulated that the DNA repair tein activation, which drives mouse double minute
mechanism becomes less efficient upon differenti- 2 homolog (Mdm2) polyubiquitination and further
ation in general. Therefore, ASCs display reduced degradation. Mdm2 is an oncoprotein that medi-
DNA damage repair (DDR) capabilities in compar- ates p53 polyubiquitination and further degrada-
ison to ESCs, which has been shown previously.26 It tion by the 26S proteasome. Therefore, Mdm2 deg-
is important to note that the mechanism of DDR in radation contributes to the increased stability of
ASCs is distinctly different than the one observed p53. It is worth noting that other mechanisms of p53
in ESCs.27 ASCs reside in a quiescent state in the G0 regulation also exist. Furthermore, p53 performs a
phase of the cell cycle. Slower cell cycle progres- regulatory function over cell proliferation by con-
sion corresponds to a higher radioresistance.28,29 trolling the expression of the p21 protein, known
Therefore, despite a lower efficacy of DDR, ASCs as cyclin-dependent kinase inhibitor.35 Silencing
exhibit a lower sensitivity to IR compared to the of p53 expression has also been shown to increase
rapidly dividing ESCs. It has been shown that the efficiency of reprogramming in iPSs genera-
upon DNA damage, ASCs can exit quiescence and tion, indicating its contribution to the maintenance
progress into the G1 phase, in which error-prone of a differentiated state.36 Nonetheless, p53 activ-
NHEJ repair is performed.30 Consequently, ASCs ity during reprogramming ensures elimination
are more susceptible to DNA damage accumula- of cells bearing genomic aberrations. Therefore,
tion, which can be passed onto progeny. disruption of p53 pathway increases the efficacy
In 1996, Schwenke et al. 17 found that -irradiation of reprogramming and the risk of mutations con-
of murine erythroid progenitor cells resulted in comitantly.37,38 Down regulation of p53 activity has
enhanced differentiation. This observed enhance- been shown to induce normal SCs transformation
ment was determined to be due to the omission towards neoplastic, tumor cells.39 This may in turn
of mitotic cell cycling, which is necessary for pro- result in cancer stem cells (CSs) formation. CSs
genitor cells to undergo terminal differentiation. share the fundamental properties of SCs, but their
Moreover, Zheng et al. 31 found that DSB suppress- activity contributes to the cancer grow and main-
es the self-renewal and promotes the further dif- tenance instead of replenishing normal cell pool.40
ferentiation of neuronal stem cells (NSCs) in a p53- Moreover, teratomas generated from p53 knockout
dependent manner. iPSs showed the presence of double-strand DNA
breaks and DDR activation, leading to the conclu-
sion that p53 inhibition decreases genomic stabil-
Role of p53 in stem cells
ity.41 Due to the high risk of tumor generation after
The p53 protein has been widely studied for many transplantation, methods utilizing p53 inhibition
years, and a number of its properties have been in iPSs generation seem to be unsuitable for thera-
elucidated.32 However, the complexity of its inter- peutic use.

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212 Mieloch AA and Suchorska WM / Radiation-enhanced stem cell differentiation

TABLE 1. The examples of adult stem cell (ASC) types and their corresponding tissue of origin, progenitors and fully differentiated cells

Organ Stem cell type Progenitors and fully differentiated cells

Bone marrow Hematopoietic stem cells Myeloid progenitor cells, Lymphoid progenitor cells

Intestine Intestinal stem cells Enterocytes, Goblet cells, Entero-endocrine cells, Paneth cells

Brain Neural stem cells Neurons, Astrocytes, Oligodendrocytes

Mammary gland Mammary stem cells Luminal cells, Myoepithelial cells

Muscle Myosatellite cells Mioblasts

that p53 is required for RA-mediated NANOG

p53 in embryonic stem cells (ESCs)
suppression.49 Therefore, synergistic cooperation
It has been shown that p53 accumulates at low levels between these two proteins may be hypothesized.
in the nucleus of hESCs, although in a deacetylated, It is important to mention that p53 also performs
inactive state. Apart from its canonical activity, p53 anti-differentiation stimulation through the Wnt
also performs a regulatory function over cell prolif- canonical signaling pathway, which is responsible
eration by controlling the expression of p21, known for the maintenance and self-renewal of human
as cyclin-dependent kinase inhibitor. p21 inhibits and murine ESCs.50,51
the activity of cyclin/cdk2 complexes and restrains
p53 in adult stem cells (ASCs)
cell cycle progression. Dolezalova et al. revealed that
after UVC-irradiation of hESCs, p21 mRNA is pre- Adult stem cells comprise endothelial progenitors
sent, although its translation is inhibited by various cells (ESC) and hematopoietic stem cells (HSC) and
microRNAs.42 However, a study by Maimets et al. tissue cells, called mesenchymal stem cells (MSC),
contradicts these findings, revealing that the small found in many different organs of the human body
molecule Nutlin, functioning as a p53 activator, el- and the one discussed in this review are listed in
evates p21 protein levels in hESCs.43 Therefore, the Table 1. Every ASC type contributes to a different
role of p21 in the p53 pathway remains elusive. cell lineage; therefore, any indications concerning
p53 plays an important role in ESC differentia- radiation-enhanced differentiation may be true for
tion. It has been shown that spontaneous differ- some ASC types and completely false for others.
entiation occurs at significantly lower rates when To clarify the reasoning behind this statement, the
the p53 level is reduced.44 However, one of the properties of p53 activity in three different types of
most crucial mechanisms supporting the theory ASCs will be described: neural stem cells (NSCs),
of radiation-enhanced differentiation is the re- hematopoietic stem cells (HSCs) and mammary
duced expression of pluripotency factors driven stem cells (MaSCs).
by p53 activity. p53 binds directly to the promot-
Neural stem cells (NSCs)
ers of NANOG and octamer-binding transcription
factor 3/4 (Oct3/4), inhibiting their transcription. Neural stem cells have the potential to differentiate
Moreover, elevated levels of p53 induce expression into neurons, astrocytes and oligodendrocytes. In
of differentiation markers GATA4 and GATA6.43 adults, neurogenesis of the central nervous system
Furthermore, upon stabilization, in addition to its begins within the subventricular zone (SVZ) and
canonical function, p53 triggers the expression of the subgranular zone of the dentate gyrus of the
miR-34a and miR-145, which subsequently repress hippocampus, which serves as a niche for NSCs.
the pluripotency factors Oct3/4, Kruppel-like fac- The SVZ is a narrow zone of tissue in the wall
tor 4 (Klf4), protein lin-28 homolog A (Lin-28A) of the lateral ventricle in the forebrain and is the
and sex determining region Y-box 2 (Sox2), which most active neurogenic region in the adult brain.52
supports differentiation.45 Neurons generated within SVZ migrate through
Retinoic acid (RA) is a commonly used differ- a path called rostral migratory stream and reach
entiation factor utilized in various differentiation their final destination within the olfactory bulb.
protocols, including those inducing the genera- A complete turn-over of resident cells within SVZ
tion of neural cells, cardiomyocytes or chondro- occurs every 2 to 4 weeks. Nearly 30 000 neuronal
cytes.4648 RA treatment results in the suppression precursors are produced daily.53
of NANOG expression. However, this effect was It has been demonstrated that the neuronal pro-
not observed after p53 gene deletion, suggesting genitors of p53-/- mice display a significantly higher

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 Mieloch AA and Suchorska WM / Radiation-enhanced stem cell differentiation 213

proliferation rate compared to wild-type mice. differentiation.60 It is also consistent with many sci-
NSCs can be maintained in culture as aggregates entific data regarding the role of p53 mutation in
or neurospheres. p53-/--derived NSCs formed sub- breast cancer development.61 Interestingly, MaSCs
stantially larger neurospheres than wild type cells, subjected to 4.0 Gy irradiation showed 2.7 fold in-
which was due to an increased number of cells per crease in mammosphere reconstitution capacity,
sphere, rather than larger cells. This finding indi- confirming that X-ray increases MaSCs prolifera-
cates that one of the functions of p53 in NSCs is to tion.62
restrain excessive proliferation.54
Hematopoietic stem cells (HSCs)
Monje et al. found that gamma irradiation of
neural progenitor cells resulted in a higher effi- Hematopoietic stem cells (HSCs) are one of the best
ciency of differentiation. Cultures irradiated with characterized human stem cells. For many years,
a 10.0 Gy dose showed increased differentiation they have been used in clinical applications, includ-
compared to cells irradiated with a 2.0 Gy dose and ing leukemia treatment. HSCs differentiate into all
control cells. However, the ratio between neurons of the blood cell lineages. They can be found in the
and astrocytes/oligodendrocytes remained undis- red bone marrow. HSCs differentiate into myeloid
turbed, which is an important factor to consider in and lymphoid progenitors, which may differentiate
the context of radioenhancement.55 further giving rise to monocytes, erythrocytes, neu-
As previously mentioned, p53 also stimulates trophils and macrophages (myeloid progenitors)
the Wnt signaling pathway. Data obtained by Wei or T-lymphocytes, B-lymphocytes and NK-cells
et al. indicated that the Wnt/-catenin signaling (lymphoid progenitors). The majority of HSCs re-
pathway plays a crucial role in the proliferation side in a quiescent state, while only a small fraction
and differentiation of NSCs in the hippocampus. In remains active and replenishes the blood cell pool.63
this study, a low dose of ionizing radiation (0.3 Gy) During steady-state hematopoiesis, p53 regu-
was shown to activate the Wnt/-catenin pathway. lates HSC self-renewal and quiescence. It is also
As a result, NSCs subjected to irradiation showed responsible for cell competition in the HSC niche.
increased proliferation and differentiation with a Cells expressing higher than average level of p53
concomitant decrease in apoptosis. Moreover, a undergo cell cycle arrest and senescence. This
water-maze test performed on mice indicated an mechanism contributes to the maintenance of tis-
improvement in the behavioral learning of these sue homeostasis by the eradication of less func-
mice after low-dose irradiation compared to non- tional cells.
irradiated mice.56 Milyavsky et al. found that HSCs subjected to
a 3.0 Gy irradiation dose exhibited a delayed DSB
Mammary stem cells (MaSCs)
repair and an increased apoptotic response via
Mammary stem cells are located in the mammary the p53/antiphagocytic protein 1 (APP1) pathway
glands. They can differentiate into all lineages of compared to progenitor cells, which indicated a
mammary epithelial cells. MaSCs are also respon- high sensitivity of HSCs to ionizing radiation.64
sible for mammary gland development during This finding is in agreement with the common no-
puberty and pregnancy.57 MaSCs can be cultured tion that HSCs are one of the cell types most vul-
in vitro as floating aggregates called mammos- nerable to ionizing radiation (IR). However, de-
pheres. A mammosphere is a spherical colony de- spite its deteriorating effects, X-ray radiation has
rived from a single MaSC by clonal proliferation.58 induced almost twofold increase in absolute num-
However, the division of MaSCs occurs predomi- ber of murine HSCs. Increased number of murine
nantly by asymmetric division. Therefore, mam- HSCs in bone marrow was still detectable 2 months
mospheres usually contain a single stem cell sur- after irradiation. This effect was not observable in
rounded by more differentiated progeny.59 Despite p21-/- mice, suggesting p21 as a key factor of X-ray
their self-renewal capabilities, they were shown to induced proliferation.62
have a limited life span in culture conditions.
MaSCs derived from p53-/- mice displayed an in-
Mitochondria in the context of enhanced
creased self-renewing potential, resulting in an in-
differentiation
creased number of MaSCs per mammosphere and
an unlimited life span in culture conditions. This Mitochondria are double-layered organelles that
finding suggests that p53 is involved in the pre- conduct the metabolic activities associated with
vention of pathological proliferation by promoting energy production through oxidative phospho-
asymmetric division, thus contributing to increased rylation. Their morphology varies between tissues

Radiol Oncol 2015; 49(3): 209-216.

214 Mieloch AA and Suchorska WM / Radiation-enhanced stem cell differentiation

TABLE 2. Examples of differences between human and murine cells affecting IR sults and a higher mutation rate.66 IR has also been
response found to induce both intracellular and mitochon-
drial oxidative stress.67 However, IR-induced mi-
Differences between human and murine DNA repair mechanisms References tochondrial production of reactive oxygen species
Murine cells are deficient in p53 global genomic repair 72 (ROS) has been proven to be the most influential in
mediating cellular damage compared to ROS gen-
Human ESC rejoin X-ray induced DSB faster than murine ESC 71
erated in other compartments.68
Murine cells repair DNA base damage more efficiently 73
Damage to mitochondria may trigger apoptosis,
Murine cells are more sensitive to oxidative stress 74,75
autophagy or, in the case of less severe lesions, fu-
Murine cells are more prone to oncogenic transformation 76,77 sion. This mechanism provides cross-complemen-
tation between impaired mitochondria, supporting
DSB = double strand breaks; ESC = embryonic stem cells their functionality by alleviation of IR-induced defi-
ciencies.69 A 0.005 to 5.0 Gy dose of X-ray radiation
has been shown to prompt a 1.5- to 3.8- fold increase
and is strictly connected to the metabolic state of a in mitochondrial mass, which supports a theory of
given cell. In addition to tissue specific differences, increased mitochondrial fusion after IR exposure.70
mitochondria may undergo fusion or fission, giv- Lan et al. have shown that ESCs subjected to IR
ing rise to tubular or fragmented mitochondria, re- display a significantly increased level of ROS gen-
spectively. The fusion/fission mechanism is strictly eration and metabolic activity.25 Both of these phe-
connected with proliferation and differentiation.65 nomena contribute to the induction of mitochon-
However, the outcome of tubular or fragmented drial fusion, which in turn is a stimulus for differ-
mitochondria generation differs between cell entiation. Therefore, it may be speculated that the
types. In ESCs, the mitochondria reside in a frag- radio-enhancement of differentiation could also in-
mented state, and an increase in mitochondrial fu- volve changes in the mitochondrial fission/fusion
sion precedes differentiation. machinery.
Ionizing radiation affects mitochondria in vari-
ous ways. Mitochondrial DNA (mtDNA) is signifi-
cantly more susceptible to IR compared to genomic Summary
DNA because it does not possess repair mecha-
nisms as efficient as those found in the nucleus. A growing amount of evidence indicates that ra-
Furthermore, mtDNA does not contain histones, diation-enhanced stem cell differentiation may be-
which results in decreased resistance to various in- come a potent tool for use in stem cell engineering

FIGURE 1. The pathway of radiation-enhanced differentiation.

Radiol Oncol 2015; 49(3): 209-216.

 Mieloch AA and Suchorska WM / Radiation-enhanced stem cell differentiation 215

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