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Epigenetic Regulation of Osteogenic and Chondrogenic Differentiation of Mesenchymal Stem Cells in Culture

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Management of mesenchymal stem cells (MSCs) capabilities to differentiate into osteogenic and chondrogenic lineages would be of utmost importance for their future use in difficult to treat cases of destroyed bone and cartilage. Thus, an understanding of the epigenetic mechanisms as important modulators of stem cell differentiation might be useful. Epigenetic mechanism refers to a process that regulates heritable and long-lasting alterations in gene expression without changing the DNA sequence. Such stable changes would be mediated by several mechanisms including DNA methylation and histone modifications. The involvement of epigenetic mechanisms during MSC bone and cartilage differentiation has been investigated during the past decade. The purpose of this review is to cover outstanding research works that have attempted to ascertain the underlying epigenetic changes of the nuclear genome during in vitro differentiation of MSCs into bone and cartilage cell lineages. Understanding such genomic alterations may assist scientists to develop and recognize reagents that are able to efficiently promote this cellular differentiation. Before summarizing the progress on epigenetic regulation of MSC bone and cartilage differentiation, a brief description will be given regarding in vitro conditions that favor MSC osteocytic and chondrocytic differentiation and the main mechanisms responsible for epigenetic regulation of differentiation.
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CELL JOURNAL(Yakhteh), Vol 15, No 1, Spring 2013 1
Review Article
Epigenetic Regulation of Osteogenic and Chondrogenic
Dierentiation of Mesenchymal Stem Cells in Culture
Mohamadreza Baghaban Eslaminejad, Ph.D.1*, Nesa Fani, M.Sc.1, Maryam Shahhoseini, Ph.D.2
1. Department of Stem Cells and Developmental Biology at Cell Science Research Center, Royan Institute for Stem Cell
Biology and Technology, ACECR, Tehran, Iran
2. Department of Genetics at Reproductive Biomedicine Research Center, Royan Institute for Reproductive Biomedicine,
ACECR, Tehran, Iran
* Corresponding Address: P.O.Box: 16635-148, Department of Stem Cells and Developmental Biology at Cell Science
Research Center, Royan Institute for Stem Cell Biology and Technology, ACECR, Tehran, Iran
Email: eslami@royaninstitute.org
Received: 4/Mar/2012, Accepted: 9/Oct/2012
Abstract
Management of mesenchymal stem cells (MSCs) capabilities to differentiate into osteo-
genic and chondrogenic lineages would be of utmost importance for their future use in
difcult to treat cases of destroyed bone and cartilage. Thus, an understanding of the
epigenetic mechanisms as important modulators of stem cell differentiation might be use-
ful. Epigenetic mechanism refers to a process that regulates heritable and long-lasting
alterations in gene expression without changing the DNA sequence. Such stable changes
would be mediated by several mechanisms including DNA methylation and histone modi-
cations. The involvement of epigenetic mechanisms during MSC bone and cartilage dif-
ferentiation has been investigated during the past decade. The purpose of this review is to
cover outstanding research works that have attempted to ascertain the underlying epige-
netic changes of the nuclear genome during in vitro differentiation of MSCs into bone and
cartilage cell lineages. Understanding such genomic alterations may assist scientists to
develop and recognize reagents that are able to efciently promote this cellular differentia-
tion. Before summarizing the progress on epigenetic regulation of MSC bone and cartilage
differentiation, a brief description will be given regarding in vitro conditions that favor MSC
osteocytic and chondrocytic differentiation and the main mechanisms responsible for epi-
genetic regulation of differentiation.
Keywords: Mesenchymal Stem Cells, Chondrogenesis, Osteogenesis, Epigenetic
Cell Journal(Yakhteh), Vol 15, No 1, Spring 2013, Pages: 1-10
Citation: Baghaban Eslaminejad MR, Fani N, Shahhoseini M. Epigenetic regulation of osteogenic and chondrogenic
differentiation of mesenchymal stem cells in culture. Cell J. 2013; 15(1): 1-10.
Introduction
Although cells of multi cellular organisms are
genetically the same, their functions and structures
differ. This diversity is due to the differential ex-
pression of genes that originate during develop-
ment and can be retained through mitosis. Such
stable alteration in gene expression is called "epi-
genetic" since they are heritable in the short term
and do not involve the mutation of DNA itself (1).
During the adult life a similar mechanism (long-
lasting changes in gene expression) occurs during
progression from stem cells into differentiated
progenies. Differentiation of stem cells into spe-
cialized cells requires an up-regulation of genes
involved in creation of a specic cell phenotype
and suppression of genes responsible for cell
stemness (2). Epigenetic regulation of stem cell
differentiation refers to the functionally relevant
modications to the genome that do not involve
changes in nucleotide sequence. Examples of such
changes are DNA methylation and histone modi-
cations (Fig 1) that, in turn, act by modifying the
accessibility of genes to transcription factors and
other modulators (3, 4).
CELL JOURNAL(Yakhteh), Vol 15, No 1, Spring 2013 2
Epigenetic Regulation of MSC Differentiation
Fig 1: Two main epigenetic modications of a genome. A. DNA methylation (Me) and B. histone modications (4).
Mesenchymal stem cells (MSCs) are adult stem
cells that possess two major properties, self-re-
newal ability and the potential for multilineage
differentiation. Although MSC have been origi-
nally isolated from bone marrow, (5, 6) further
investigation has shown that multiple tissues con-
tain MSC-like populations (7-16). Reportedly, the
most important characteristics of MSCs are their
potential for differentiation into bone and cartilage
cell lineages (5, 6). This capacity has generated
tremendous excitement for the regeneration of
damaged bone and cartilage tissues that are either
incurable or difcult to cure due to insufciency
or failure of current therapies (17-20). Generally,
there two strategies for the application of MSCs
in regenerative medicine. One strategy uses cells
in an undifferentiated state, which allows them to
undergo differentiation atthe defective site. The
disadvantage of this strategy is the unwanted dif-
ferentiation of cells at the repair site. For instance,
if MSCs are to be used for the regeneration of
cartilage tissue, bone cells may be produced by
unwanted cell differentiation. An alternative ap-
proach is to fully differentiate MSCs into the de-
sired cells prior to their transplantation (21, 22).
With this strategy, the in vitro differentiation of
MSCs into bone and cartilage cell lineages seems
to be an inevitable step prior to their application in
the cell-based treatment of tissue defects. There-
fore, the differentiation process of MSCs must be
thoroughly understood, particularly in terms of its
regulatory mechanisms.
From the discovery of MSCs until now, numerous
attempts have been made to understand their differ-
entiation process. Particularly, research has focused
on differentiation into bone and cartilage cell lin-
eages the in vitro conditions favoring MSC bone
and cartilage differentiation. Furthermore, gene ex-
pression prole during progression from stem cell
into bone and cartilage cells are mostly revealed
(reviewed below). Another issue related to MSC
differentiation is the epigenetic regulation underly-
ing their osteocytic and chondrocytic differentiation
of which investigations have recently begun. The
purpose of this paper is to briey review the main
epigenetic mechanisms including DNA methylation
and histone modications, to summarize all studies
that have attempted to determine the underlying
epigenetic changes of the nuclear genome during
MSC bone and cartilage differentiation, and nally
to highlight the importance of epigenetic studies
in bone and cartilage engineering and regenerative
medicine. First, a brief description will be given re-
garding in vitro conditions necessary for osteocytic
and chondrocytic differentiation of MSCs and the
main transcription factors that promote tissue-spe-
cic gene expression during differentiation.
In vitro bone differentiation
In vitro bone differentiation of MSCs is a com-
plex process requiring multiple soluble inducers.
To establish an osteogenic culture, a conuent
AB
CELL JOURNAL(Yakhteh), Vol 15, No 1, Spring 2013 3
Baghaban Eslaminejad et al.
monolayer culture of MSCs must be prepared and
provided with osteogenic medium, which typical-
ly consists of a basal medium such as Dulbecco’s
modied eagle medium (DMEM) supplemented
with osteogenic inducers. The most-frequently
used osteogenic supplement is composed of dexa-
methasone (10 nM), ascorbic acid (50µg/ml) and
ß-glycerol phosphate (10 mM). Dexamethasone is
the essential component; its continual supplemen-
tation is required for human MSC ostegenic differ-
entiation (23). Ascorbic acid, another osteogenic
component, is not essential for MSC bone differ-
entiation but its addition enhances production of
collagen-rich extracellular matrix (ECM) (24). ß-
glycerol phosphate in the osteogenic medium pro-
vides favorable conditions for culture mineraliza-
tion (25, 26).
In addition to the above mentioned frequent-
ly used reagents, other factors that impact MSC
differentiation into a bone cell lineage include 1,
25-dihydroxyvitamin D3 (27) and estrogen (28).
According to some studies parathyroid hormone
(PTH) exhibits an osteogenic effect on MSCs
if the culture is exposed intermittently to PTH
(29, 30). Local factors including prostagland
in,transforming growth factor-beta (TGF-β), -
broblast growth factor-2 (FGF-2) and bone mor-
phogenetic proteins (BMPs), particularly BMP6,
have been reported to promote in vitro MSC osteo-
genesis (31-33). Other factors which have osteo-
genic effects include lithium chloride (LiCl) and
6-bromoindirubin-3΄-oxim (BIO) (33). Addition-
ally, melatonin, a hormone secreted by the pineal
gland exhibits osteogenic effects on MSC culture
(34). The osteogenic factors thus far mentioned are
more effective when used synergistically. For ex-
ample, it has been shown that addition of BMP2
into a rat MSC culture enhanced the osteogenic
potency of FGF-2. Dexamethasone and vitamin
D3 as well as BMP2 and retinoic acid have been
shown to exhibit a synergistic effect on MSC os-
teogenic culture (35-37).
Osteogenic supplements of the MSC monolayer
culture eventually lead to expression of specic
osteoblastic transcription factors. Core binding
factor alpha 1 (Cbfa1), which is also called Runx2,
is one of the most studied transcription factors ex-
pressed in MSCs upon their commitment toward
an osteogenic differentiation (38, 39). Upon ex-
pression, Runx2 must be activated through post-
translational modications or protein-protein
interactions (40). Other transcription factors
may collaborate with Runx2 to promote os-
teogenic differentiation. It has been found that
TAZ, a transcriptional co-activator, co-activates
Runx2-dependent gene transcription in murine
MSCs (41). Runx2 activates the expression of
bone-related genes, including osteocalcin, col-
lagen I, osteopontin, bone sialo protein and the
parathormon receptor (PTHR) (39).
Osterix is another transcription factor whose in-
volvement has been discovered in MSC bone dif-
ferentiation. This discovery was particularly nota-
ble in murine MSCs transduced with the osterix
gene (42).
In vitro cartilage differentiation
The induction of chondrogenesis in MSCs de-
pends on the coordinated activities of two funda-
mental parameters: cell density and growth fac-
tors (43-46). The TGF-β super family of proteins
and their members, such as BMPs are established
regulatory factors in chondrogenesis. TGF-β pro-
motes proteoglycan deposition, so that in its ab-
sence the ECM of differentiated cells contains
modest amounts of proteoglycan (47). TGF-β1 is
a standard media additive used in cultures to in-
duce chondrogenesis. TGF-β3 has been shown to
induce a more rapid, representative expression of
a chondrogenic culture (48, 49). In the cell labora-
tory, cartilage differentiation of MSCs can be per-
formed in a pellet culture system. Approximately
2 × 105 cells (passages 2-3) must be condensed in
to a pellet by centrifugation at 300 g for 4 minutes,
followed by incubation in an atmosphere of 37˚C
and 5% CO2 in a 0.5 ml chondrogenic medium.
The chondrogenic medium should be composed
of 10 ng/ml TGF-ß3, 500 ng/ml BMP-6, 100 nM
dexamethasone, 50 µg/ml ascorbic 2-phosphate,
50 µg/ml ITS and 1.25 mg/ml bovine serum al-
bumin. Recently, we have shown that addition of
Lithium Chloride and a small molecule refereed
to as SB216763 can enhance glycoseaminogly-
cal deposition in the human marrow-derived MSC
chondrogenic culture (50).
Sox9 is the main transcription factor essential for
chondrocyte differentiation of MSCs. In the chon-
CELL JOURNAL(Yakhteh), Vol 15, No 1, Spring 2013 4
Epigenetic Regulation of MSC Differentiation
drogenic culture of MSCs. Expression of Sox9 is
followed by chondrocyte-specic gene expression
that includes collagen I and aggrecan. Genetic mu-
tations in Sox9 leads to congenital dwarsm syn-
drome (51).
Epigenetic mechanisms
DNA methylation
Currently, one of the epigenetic changes mostly
studied in mammals is DNA methylation, which
primarily involves the establishment of paren-
tal-specific imprinting during gametogenesis
(52). This process includes covalent binding
of a methyl group from a methyl donor, mainly
S-adenosylmethionine, to carbon 5 of the cyto-
sine that often is located in the CpG sites. This
enzymatic reaction is produced by a family of
enzymes called DNA methyltransferases (Dn-
mts) (53). There are several types of Dnmts, in-
cluding de novo Dnmt3a and Dnmt3b,which are
highly expressed in the developing mouse em-
bryo and promote global de novo methylation
after implantation (54). Dnmt1 is a methyltrans-
ferase that maintains the existing methylation
patterns upon cell division (52). Genomic re-
gions that contain a high number of methylated
cytosine are usually transcriptionally inactive.
The absence of DNA methylation is a prerequi-
site for transcriptionally active genes (55, 56).
Histone modications
Histones, the major structural proteins of chro-
mosomes, are small proteins that contain numer-
ous positively-charged amino acids such as lysine
and arginine. These positively charged amino acids
enable histones to tightly bind with the phosphate-
sugar backbone of double stranded DNA. These
proteins have a tail comprised of a long aminoacid
chain in their N-terminal domain that plays an im-
portant role in regulation of chromatin structure.
The histone tail domains are considered as master
control switches that dene the structural and func-
tional characteristics of chromatin at many lev-
els. These structures modulate DNA accessibility
within the nucleosome and are essential for stable
folding of oligonucleosome arrays into condensed
chromatin bers (57). Histone tails may have vary-
ing fates including acetylation, methylation, phos-
phorylation, polyadenylation, ribosylation, ubiqui-
tination and glycosylation. Combinations of these
modications determine the overall interaction of
histones with the DNA molecule, leading to acti-
vation and/or inhibition of transcription (58). Of
these, acetylation and methylation are the moste-
pigenetic mechanisms studied in transcriptional
regulation.
Acetylation is one of the studied histone modica-
tions that occurs primarily at the lysine of histones
3 and 4, and is basically catalyzed by acetyltrans-
ferase enzymes such as HBO1, TIP60, MORF/Moz
and MOF. The consequence of this modication
is the loss of the positive charge of the lysine resi-
due which affects the histone’s binding to the DNA
molecule,and is dened as nucleosome opening
(Fig 2). Acetylation levels of histone tails depend-
ent on balance between the two enzymatic activities
of acetyltransferase and deacetylase (58). There are
four classes of histone deacetylase (HDAC). Class I
includes HDAC 1, 2, 3, and 4. Class II is comprised
of HDAC5, 6, 7, 9, and 10. Class III includes Sirtuin
1-7 and class IV includes HDAC11. Among these,
the HDAC of classes I, II and IV have the same se-
quences and structures. Sirtuin, however, has a dif-
ferent structure and a different catalytic mechanism.
Sirtuin proteins comprise a unique class of NAD ±
dependent protein deacetylases (59).
Fig 2: Histone acetylation and deactylation. Histone acetyl-
transferase (HATs) adds acetyl groups (Ac) onto histone
tails,which results in a nucleosome openingthus allowing
for transcription factors to access DNA and initiate gene
transcription.Histone deacetylases (HDACs) remove the Ac
from the histone tails, leading to a closed chromatin struc-
ture (61).
CELL JOURNAL(Yakhteh), Vol 15, No 1, Spring 2013 5
Baghaban Eslaminejad et al.
Acetylation of the histone tails leads to neu-
tralization of the partial electric charge of lysine
which in turn results in opening of the chromatin
structure. In vitro observation of this event is not
a simple task, but biophysical analysis has shown
that intranuclosomal linkages are important for
chromatin stabilization. According to research,
acetylation of lysine 9 on histone 3 has a dominant
negative effect on the formation of 30 nanometer
chromatin bers and higher-order structures (60).
Methylation is another histone modication that
plays different roles in epigenetic regulation of
gene expression. Histonemethylation usually oc-
curs on lysine and argenine residues (58). Among
these, methylation of lysines 4, 9, 27, 36, and 79 in
histone 3 and lysine 20 in histone 4 are dened in
transcriptional regulation. Methylation of lysines 9
and 27 of histone 3 (H3-K9, H3-K27) are present in
silent chromatin domains which are mainly related
to heterochromatin regions and inactive promot-
ers. In contrast, methylation of lysine 4 of histone
3 (H3-K4) which activates chromatin is primarily
observed in active promoters. If these two modi-
cations simultaneously occur in a promoter region,
the relevant gene goes to a poised state. This dual
epigenetic mark is observed in pluripotent cells
during development. The consequence of this "bi-
valent mark" is inactivation of the genes respon-
sible for specic cellular differentiation (62, 63).
Lysine can be mono-, di-or tri-methylated but ar-
gentine can be only mono-methylated.The level of
histone methylation is controlled by the dual enzy-
matic activities of methyl transferase and demeth-
ylase (64). Basically, there are two classes of pro-
teins that include thepolycomb group and tritorax
group complexes which act as methyl transferase
elements during development. These histone meth-
ylating enzymes encode methylation of lysine 27
and lysine 4 of histone 3, respectively. It has been
shown that a precise balance between these two
enzymatic activities modulates epigenetic regula-
tion of cellular differentiation processes (58).
Epigenetics of bone differentiation
Over the past decade, several researchers have
investigated epigenetic control of MSC bone dif-
ferentiation. In this context and according to nu-
merous research DNA methylation is dynamically
involved in the process of bone differentiation of
MSCs. For example, Villagra et al. have observed
a signicant hypermethylation at the osteocalcin
gene locus in undifferentiated cells, which was as-
sociated with the condensed chromatin structure.
Their subsequent examination has revealed that
during in vitro osteoblast differentiation, CpG
methylation of the osteocalcin promoter signi-
cantly decreased as the osteocalcin geneu pregu-
lated (65).
Arnsdorf et al. have designed a novel protocol to
promote MSC osteogenic differentiation by the ap-
plication of a mechanical stimulus. Following suc-
cessful differentiation they attempted to determine
the possible underlying mechanism of MSC osteo-
genesis. According to their results, the increase ob-
served in bone-specic gene expression was under
the control of epigenetic regulation of several os-
teogenic candidate genes. Mechanical stimulation
of MSCs reduced the DNA methylation state of the
genes, which lead to their increased expression (66).
Involvement of DNA methylation in osteogenic
differentiation of MSCs has also been reported by
Dansranjavin et al. (67). They demonstrated that
differentiation of MSCs into osteoblast and adi-
pocyte cells was accompanied by reduced expres-
sion of the stemness genes such as Brachyury and
LIN28, which basically occurred via hypermeth-
ylation of their promoter regions (67).
Hsiao et al. have observed epigenetic regula-
tion of the thyroid hormone receptor interactor 10
(Trip 10) during osteogenic induction of human
bone marrow-derived MSCs. To determine wheth-
er DNA methylation-induced gene silencing was
involved in this process, they applied an in vitro
method that specically methylated the Trip 10
promoter. The transfection of exogenous methyl-
ated Trip 10 promoter DNA into MSCs resulted in
progressive accumulation of methyl-cytosines at
the endogenous Trip 10 promoter, reduced Trip 10
expression, and accelerated MSC-to neuron and
MSC-to-osteocyte differentiation (68).
In contrast, Kang et al. have reported the lack
of a notable change in methylation levels of the
promoter region after in vitro osteocytic differen-
tiation of MSCs (69).
Histone acetylation is another epigenetic mecha-
nism reported to be involved in osteogenesis. Shen
et al. have investigated the chromatin-mediated
CELL JOURNAL(Yakhteh), Vol 15, No 1, Spring 2013 6
Epigenetic Regulation of MSC Differentiation
mechanisms by which the bone-specific osteo-
calcin gene is transcriptionally activated during
cessation of cell growth in ROS 17/2.8 osteosar-
coma cells, as well as during normal osteoblast
differentiation (70). They assayed acetylation of
histones H3 and H4 at the osteocalcin gene pro-
moter during and after cell proliferation by us-
ing the chromatin immunoprecipitation (ChIP)
technique. These researchers observed that both
the promoter and coding region of the osteoc-
alcin gene contained high levels of acetylated
H3 and H4 histones during the proliferative
period of osteoblast differentiation. According
to their findings active expression of the oste-
ocalcin gene in mature osteoblast and conflu-
ent ROS 17/2.8 cells is functionally linked to
preferential acetylation of core histones (70).
In contrast, Tan et al. have used microarrays to
investigate the roles of histone modifications
(H3K9Ac and H3K9Me2) upon the induction
of human MSC osteogenic differentiation. In
their research, enrichment of H3K9Acglobally
decreased at the gene promoters whereas the
number of promoters enriched with H3K9Me2
increased upon bone differentiation (71). We
have attributed the discrepancies in these two
reports to the difference in the cells (cell line
or MSCs) and the method (ChIP or microarray)
used in each experimental design.
Others, however in order to study the reverse role
of histone deacetylation in osteogenes is preferred
to measure the acetylation/deacetylation process.
Lee et al. examined the expression level of HDAC
and degree of histone acetylation at the promoter
regions of osteoblast genes. They have noted that
down-regulation of HDAC1 is an important pro-
cess for osteogenesis (72).
Histone methylation has also been reported as
an epigenetic mechanism underlying MSC oste-
ogenic differentiation. In this context Hassan et
al .have found that HOXA10 (a gene necessary
for embryonic patterning of skeletal elements)
contributes to osteogenic lineage determination
through activation of Runx2, alkaline phos-
phatase, osteocalcin and bone sialoprotein (73).
Their further investigations have revealed that
these effects are mediated through total chro-
matin hyperacetylation and H3K4 hypermeth-
ylation of the genes. In this context, Fan et al.
have found that the BCL-6 corepressor (BCOR)
mutation increases histone H3K4 and H3K36
methylation in MSCs. This, in turn, reactivates
transcription of the osteo-dentinogenic gene in
MSCs. In their study MSCs were isolated from a
patient with oculo-facio-cardio-dental (OFCD)
syndrome which is the result of a mutation in
the BCOR gene. This syndrome is characterized
by canine teeth with extremely long roots, con-
genital cataracts, craniofacial defects, and con-
genital heart disease (74).
Involvement of histone methylation in MSC
bone differentiation is also supported by the work
of Wei et al. These authors have found that the
activation of cyclin-dependent kinase 1 (CDK1)
promotes MSC bone differentiation through phos-
phorylization of theenhancer of the zeste homo-
logue 2 (EZH2) which is the catalaytic subunit of
the polycomb repressive complex 2 (PRC2) that
catalizes trimethylation of histone H3 on Lys 27
(H3K27) at Thr 487 (75).
Thus, according to the above-mentioned stud-
ies, several epigenetic regulations that include
DNA methylation, histone acetylation and
methylation might involve MSC osteogenic
differentiation. It is not clear whether all three
mechanisms are simultaneously involved dur-
ing MSC bone differentiation or if only one
mechanism promotes differentiation dependent
on the culture conditions. This issue needs ad-
ditional investigation.
Epigenetics of cartilage differentiation
Few studies have been conducted with regards
to epigenetic regulation of gene expression dur-
ing MSC cartilage differentiation. The work by
Ezura et al. (76) isnotable. These authors have in-
vestigated the CpG methylation status in human
synovium-derived MSCs during in vitro chondro-
genesis and found that DNA methylation levels of
CpG-rich promoters of chondrocyte-specic genes
were mostly maintained at low levels (76).
There are many investigations in which the epi-
genetic mechanism involved in cartilage differen-
tiation has been investigated by the use of chon-
drocyte or relevant cell lines. Histone acetylation
is among theepigenetic mechanisms that have
been reported to be involved in cartilage-specic
CELL JOURNAL(Yakhteh), Vol 15, No 1, Spring 2013 7
Baghaban Eslaminejad et al.
gene expression. In this context the role of p300,
an enzyme possessing a histone acetyltransferase
(HAT) activity, was observed in several studies.
Using the chondrosarcoma cell line SW1353,
Tsuda et al. have shown that Sox9 associates with
CREB-binding protein (CBP)/p300 via its car-
boxyl termini activation domain and functions as
an activator for cartilage tissue-specic gene ex-
pression during chondrocyte differentiation (77).
Later, Furumatsu et al. have investigated the mo-
lecular mechanism of synergy between Sox9 and
p300 in chromatin mediated transcription on chro-
matinized templates in vitro. Their results revealed
that p300 potentiated Sox9-dependent transcrip-
tion through hyperacetylation of histones. P300/
CBP acts as a coactivator to cartilage homeopro-
tein-1 (Cart1) through acetylation of the conserved
lysine residue adjacent to the homeodomain (78).
This point has been mentioned by Iioka et al. who
have conducted a study using an in vitro acetyla-
tion assaythat investigated the functional involve-
ment of p300/CBP during chondrogenesis. Cart1
is expressed selectively in chondrocyte lineage
during embryonic development (79).
Histone deacetylation by HDAC1 has been re-
ported to have a critical inhibitory role in cartilage
noncollagenous matrix deposition during cartilage
differentiation. Cartilage oligomeric matrix pro-
tein (COMP) is a noncollagenous matrix protein in
cartilage. In a study using Sox-9-null mice, Liu et
al. in 2007 have shown that the COMP gene was
inhibited by a transcription repressor,the negative
regulatory element (NRE)-binding protein by re-
cruiting HDAC1 to the COMP promoter (80). In
another study by the same authors on rat chon-
drosarcoma cells and BMP-2-treated C3H10T1/2
progenitor cells, it was observed that the leuke-
mia/lymphoma-related factor, a POZ domain-con-
taining transcriptional repressor, interacted with
HDAC1 and inhibited COMP gene expression and
chondrogenesis (81).
Using HDAC4-null mice, Vega et al. have found
that HDAC4 regulates chondrocyte hypertrophy
and endochondral bone formation by inhibiting the
activity of Runx2 which is a transcription factor
necessary for chondrocyte hypertrophy. It has been
shown that HDAC4-null mice display premature
ossication of developing bone; and conversely,
over expression of HDAC4 in proliferating chon-
drocytes in vivo inhibits chondrocyte hypertrophy
and differentiation (82).
In contrast to deacetylation, histone acetylation
favors cartilage differentiation which has been
shown in both in vivo and in vitro studies conduct-
ed by Hattori et al. These authors have conducted a
study to determine Sox9-regulated gene transcrip-
tion during chondrogenesis. In this study, they
have found a specic interaction between Sox9
and Tat interactive protein-60 (Tip60) which leads
to enhanced acetylation of Sox9, mainly through
the K61, 253, and 398 residues and subsequent en-
hancement of its transcriptional activity (83).
In some studies, results have shown that activity
of HDAC in cartilage differentiation is mediated
through the Wnt signaling pathway. In this context
Huh et al. have investigated the role of HDAC in
the expression of type II collagen that is a marker
of differentiated chondrocytes. They have found
that HDAC activity in a primary culture of articu-
lar cartilage decreased during dedifferentiation
that had been induced by serial monolayer culture;
the activity was recovered during 3-D culture. It
was also observed that HDAC inhibition promoted
the expression of Wnt-5a which is known to inhib-
it type II collagen expression. Conversely, knock-
down of Wnt-5a blocked the ability of HDAC in-
hibitors to suppress collagen II expression. They
have concluded that HDAC promotes collagen
II expression by suppressing the transcription of
Wnt-5a (84).
In conjunction and according to a study on MSCs,
during chondrogenic differentiation DNA methyla-
tion levels of CpG-rich promoters of the chondro-
cyte-specic genes are mostly maintained at low
levels. Conicting reports exist for non-MSCs, how-
ever numerous studies have reported an association
between histonehyperacetylation and chondrogenic
differentiation, (78, 79) or the inhibition of carti-
lage differentiation by histone deacetylation (80-83).
Some researchersbelieve that cartilage differentiation
is associated with histone deacetylation (84).For fur-
ther clarication of the subject, additional research
must be performed using MSCs.
Application of epigenetics in bone and cartilage
engineering and regeneration
The knowledge obtained by epigenetic studies
CELL JOURNAL(Yakhteh), Vol 15, No 1, Spring 2013 8
Epigenetic Regulation of MSC Differentiation
on MSC osteocytic/chondrocytic differentiation
could be applied to bone and cartilage engineering
as well as regenerative medicine. As mentioned
earlier, epigenetic modication is the process of
adding and removing chemical tags,i.e. acetyl or
methyl groups, on DNA or its surrounding his-
tones which results in activation or suppression of
the genes involved in stem cell differentiation. On
the other hand the key process in MSC-based bone
and cartilage engineering is to efciently direct the
cells into differentiated phenotypes within an ap-
propriated 3-D scaffold. After identication of epi-
genetic tags underlying MSC bone and cartilage
differentiation, the next step would be to locate
suitable chemicals or pharmaceuticals that are able
to promote those epigenetic modications. By us-
ing these reagents appropriate bone and cartilage
constructs could be developed. Such constructs
could be used for transplantation into large bone
and cartilage defects which are considered to be
problematic in the eld of orthopedics.
Conclusion
MSCs are considered as promising cell can-
didates for future treatment of difcult bone and
cartilage defects. Some scientists believe that
transplantation of MSCs at the differentiated state
would be more advantageous than transplantation
at the undifferentiated state. Thus, investigations
of MSC osteogenic and chondrogenic differentia-
tion are of utmost importance. One objective of
this research would be to dene the precise con-
dition under which MSC differentiation can occur
in a controlled, predictable manner. Understand-
ing epigenetic control of cell differentiation will
certainly enable scientists to achieve this goal. In
this context, promising progress has been made af-
ter approximately a decade of research. It has been
revealed that DNA methylation, as well as histone
acetylation and methylation are involved in MSC
bone differentiation.
In the context of cartilage differentiation of
MSCs, to the best of our knowledge, there are few
studies that have been performed. Most have been
conducted using chondrocytic cells or related cell
lines. According to these, predominantly DNA
methylation and histone acetylation are involved
in the control of cartilage differentiation. Under-
standing the epigenetic mechanism that regulates
cell differentiation may result in the development
of an appropriate reagent or enzyme that could
promote the necessary epigenetic changes of the
genome required for efcient differentiation of
MSCs. This, in turn, would be considered the pref-
erential cellular material with which to regenerate
large defects in bones and cartilages.
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... Histone deacetylases (HDACs) revert the process by removing the acetyl groups. (Modified from (Eslaminejad et al., 2013)). ...
Thesis
Neurons have a highly specialized morphology whose signal-regulated remodeling is key to their function in neuronal networks. Activity-dependent nuclear calcium signaling is a crucial regulator of gene transcription in hippocampal and spinal cord neurons and mediates gene expression by directly acting on transcription factors or by regulating epigenetic processes, such as the induction of DNA methyltransferases (DNMTs) or the nucleo-cytoplasmic shuttling of class IIa histone deacetylases(HDAC4, -5, -7, and -9). Epigenetic mechanisms regulate several neuroadaptive phenomena in hippocampal neurons including, among others, synaptic plasticity and memory formation. In the first part of this thesis we describe how the subcellular localization of HDAC4 controls the morphology of hippocampal neurons by modulating the expression of a factor critical for dendrite architecture. Epigenetic regulators have also been suggested to mediate central sensitization in spinal cord neurons and the development of chronic pain. The transition from acute to chronic pain is considered a pathological manifestation of neuronal plasticity in nociceptive pathways and shares common molecular pathways with memory formation. However, if and how epigenetic gene regulatory events control also structural remodeling of spinal cord circuits, relevant for central sensitization, remains to be investigated. Here we characterized the impact of synaptic activity and chronic inflammatory pain on the expression and activity of DNMTs and HDACs in spinal cord neurons and found that the de novo methyltransferase Dnmt3a2 and HDAC4 were particularly affected. We demonstrate that activity-induced levels of Dnmt3a2 contribute to spinal sensitization and hypersensitivity in the CFA model of inflammatory pain by regulating the expression of pain- and plasticity-related genes. Moreover, we found that long-lasting, but not acute inflammatory pain, results in a nuclear export of HDAC4 in spinal cord neurons, which is accompanied by increased levels of histone 3 acetylation. Using recombinant adeno-associated virus-mediated expression of a nuclear localized dominant active mutant of HDAC4 in dorsal horn neurons, we demonstrated that nuclear HDAC4 blunts the development of mechanical hypersensitivity without affecting acute nociception. Next generation RNA-sequencing analysis produced a list of HDAC4-regulated candidate genes in the context of chronic inflammatory pain. The identified candidates include both well-known and novel mediators of chronic pain development and have been functionally tested in vivo with gain of function and loss of function experiments. Our results identify HDAC4 and its target genes, as key epigenetic regulators of central sensitization in chronic inflammatory pain and as possible targets for pain therapies.
... 18 In addition, epigenetic mechanisms, through the DNA modifying enzymes DNMTs and Ten-Eleven-Translocation (TETs), play an important role in the transcriptional control of genes related to both maintenance of pluripotentiality 19,20 and osteogenic potential. 21,22 Thus, the objective of this study was to determine the impact of the individual epigenetic and transcriptional profiles on the osteogenic potential of PDLCs, evaluating DNA methylation as a mechanism of transcriptional control. ...
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Background: Mesenchymal cells' biology has been an important investigative tool to maximize bone regeneration through tissue engineering. Here we used mesenchymal cells from periodontal ligament (PDLCs) with high (h-) and low (l-) osteogenic potential, isolated from different donors, to investigate the impact of the individual epigenetic and transcriptional profiles on the osteogenic potential. Materials and methods: Genome-wide and gene-specific DNA (hydroxy)methylation, mRNA expression and immunofluorescence analysis were carried out in h- and l-PDLCs at DMEM (non-induced to osteogenesis) and OM (induced - 3rd and 10th days of osteogenic differentiation) groups in vitro. Results: Genome-wide results showed distinct epigenetic profile among PDLCs with most of the differences on 10th day of OM; DMEMs showed higher concentrations (xOM) of differentially methylated probes in gene body, intronic and open sea (3rd day), increasing this concentration in TSS200 and island regions, at 10 days. At basal levels, h- and l-PDLCs showed different transcriptional profiles; l-PDLCs demonstrated higher levels of NANOG/OCT4/SOX2, BAPX1, DNMT3A, TET1/3 and lower levels of RUNX2 transcripts, confirmed by NANOG/OCT4 and RUNX2 immunofluorescence. After osteogenic induction, the distinct transcriptional profile of multipotentiality genes was maintained among PDLCs. In l-PDLCs, the anti-correlation between DNA methylation and gene expression in RUNX2 and NANOG indicates methylation could play a role in modulating both transcripts. Conclusions: Epigenetic and transcriptional distinct profiles detected at basal levels among PDLCs were maintained after osteogenic induction. We cannot discard the existence of a complex that represses osteogenesis, suggesting the individual donors' characteristics have significant impact on the osteogenic phenotype acquisition. This article is protected by copyright. All rights reserved.
... Scaffold enhancement has become a widespread method to incorporate different factors that can more fully mimic the physiological function and architecture of bone tissue. Given the promise of EVs in therapeutic applications, it is important to recognize the epigenetic roots that underlie bone and cartilage degeneration (Van Meurs et al., 2019) and the potential use of chemicals and pharmaceuticals to promote the desired epigenetic modifications that drive chondrogenic and osteogenic differentiation in scaffolds (Eslaminejad et al., 2013). Indeed, EVs have potential to serve as vehicles for drug and molecule delivery to support skeletal tissue regeneration. ...
... Histone acetylation on lysine residues is tightly regulated by opposing actions of two families of enzymes, histone acetyltransferases (HATs) and histone deacetylases (HDACs) [40]. Hyperacetylation of histone tails induced by HATs results in an open chromatin formation that usually correlates with gene activation, whereas deacetylation by HDACs mediate a closed chromatin conformation and transcriptional suppression [41]. To further explore the presence of post-translational histone modifications in the regressing interdigits, we analyzed the expression of HDACs genes in the developing autopod by in situ hybridization (Figure 3). ...
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... hBMSCs are typical adult MSCs with self-renew capabilities and can differentiate into osteoblasts, which may be a potential cell source for bone tissue engineering 30 . The osteogenic differentiation of hBMSCs is vital for the application for bone regeneration 31 . Therefore, it is important to study the molecular mechanisms that control hBMSCs osteogenic differentiation. ...
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