Journal of Cellular Biochemistry 97:744–754 (2006)
Expansion of Mesenchymal Stem Cells Isolated From
Pediatric and Adult Donor Bone Marrow
Katia Mareschi,1* Ivana Ferrero,1Deborah Rustichelli,1Simona Aschero,1Loretta Gammaitoni,2
Massimo Aglietta,2Enrico Madon,1and Franca Fagioli1
1Department of Pediatrics, Regina Margherita Children’s Hospital, University of Turin, Turin, Italy
2Department of Oncological Sciences, Institution of Cancer Research and Treatment, IRCC Candiolo,
marrow (BM) of pediatric and young adult donors, to analyze the growth kinetic, immunophenotype, telomere length,
expression in the MSCs isolated from two groups until 10th passage (77 days) and there was no significant difference
between the modulation of antigen expression. In particular, at the first passage, MSCs showed a low contamination of
hemopoietic cells which became insignificant in the following passages. There was a high expression of CD90, CD29,
CD44 and CD105 and variable and moderate expression of CD166 and CD106 at the start of MSC culture and at each
passage during expansion. No chromosomal alteration or evidence of cellular senescence were observed in all analyzed
samples. All these data suggest that MSCs can be isolated and expanded from most healthy donors, providing for an
autologous source of stem cells. J. Cell. Biochem. 97: 744–754, 2006.
The enormous plasticity of mesenchymal stem cells (MSCs) suggests an improvement of a standard
? 2005 Wiley-Liss, Inc.
Key words: mesenchymal stem cells; expansion; pediatric donor; adult donor
Bone marrow (BM) contains two different
stem cell populations: those of the hemopoietic
lineage, hemopoietic stem cells (HSCs), which
reconstitute the hemopoietic system with all
(MSCs), mesoderm precursors which normally
differentiate into multiple mesodermal tissue,
including: bone [Haynesworth et al., 1996];
cartilage [Yoo et al., 1998]; adipose [Park et al.,
1999]; muscle [Wakitani et al., 1995]; tendon
[Awad et al., 1999]; stroma [Majumdar et al.,
1998]; and neuron cells overcoming their germ-
inal commitment [Kopen et al., 1999; Brazelton
et al., 2000; Black and Woodbury, 2001; Kim
et al., 2002; Woodbury et al., 2002].
MSCs are easily isolated from BM as they can
adhere to plastic and are capable of substantial
proliferation and expansion in culture [Pitten-
ger et al., 1999]. Undifferentiated MSCs exhibit
a fibroblastic like morphology and a character-
istic pattern of cell-surface antigens maintain-
ing the ability to differentiate into multiple
cell types, establishing their stem cell nature
expanded in vitro and cryopreserved with no
loss of phenotype or differentiation potential
over, MSCs produce several cytokines, growth
factors, and cell adhesion molecules; important
factors which influence the hemopoietic micro-
environment [Eaves et al., 1991; Haynesworth
et al., 1992; Majumdar et al., 1998]. They
constitute an important component of the my-
elosupportive stroma which is damaged by the
conditioning regimen for stem cell transplanta-
tion and preliminary results suggest that co-
transplantation with expanded MSCs resulted
? 2005 Wiley-Liss, Inc.
*Correspondence to: Katia Mareschi, Department of Pedia-
trics, University of Turin, P.za Polonia 94, 10126 Turin,
Italy. E-mail: email@example.com
Received 8 August 2005; Accepted 9 September 2005
in faster engraftment and a reduction in the
incidence and severity of graft-versus-host in
some patients [Lazarus et al., 2005].
Ex vivo expanded cells undergo several divi-
sions which might induce excessive cellular
senescence, therefore determination of the
aging is relevant before an extensive clinical
application of ex vivo manipulated MSCs
[Hodes, 1999]. Progressive shortening of telo-
mere length has been suggested as acting as a
mitotic clock which may contribute to cellular
senescence and as a good predictor of the
remaining replicative capacity of the somatic
cells [Vaziri et al., 1994].
Previous studies have shown that human
MSCs expanded in vitro tend to lose their proli-
ferative potential, homing capacity, and in vivo
bone forming efficiency aging [Mets and Ver-
donk, 1981; Digirolamo et al., 1999; D’Ippolito
et al., 1999; Banfi et al., 2000; Rombouts and
Ploemacher, 2003; Stenderup et al., 2003].
From these observations it is clear that
different variables and parameters must be
considered in the isolation and expansion of
MSCs for experimental and clinical use.
Several studies were performed on the age-
related effect on the number, cellular growth,
cells identified as MSC or stromal cells. Some
studies reported an age-related decrease in the
number of osteoprogenitor cells, while other
studies showed no effects or an age-related
Tsuji et al., 1990; Egrise et al., 1992; Quarto
et al., 1995; Bergman et al., 1996; Oreffo et al.,
Stenderup et al., 2001]. Stenderup et al. 
demonstrated that aging is associated with a
decreased proliferate capacity of MSCs but not
with a function capacity. Recently, Baxter et al.
 reported the effect of in vitro expansion
on the replicative capacity of MSCs isolated
from pediatric and adult (59–75 years aged)
donors on the basis of the telomere loss during
the in vitro expansion. These studies were all
performed on MSCs isolated from adult donors,
which were divided into young and old (over
50 years of age) adult donors.
In this study, we isolated and analyzed MSCs
from healthy donor bone marrow (BM) to
analyze the modulation of the immunopheno-
typic characteristics, telomere length, and
karyotype modifications during their cellular
expansion and to verify whether MSCs isolated
from pediatric and young adult donors showed
MATERIALS AND METHODS
Harvest and Preparation of MSCs
adultorpediatricCaucasian donors whounder-
went BM collection for a related patient after
anunfiltered BM collection bag(BaxterHealth-
care Corporation, IL) which was normally
discarded before the BM infusion. The bag was
washed three times with phosphate buffer
saline (PBS) 1? (Cambrex Bioscience, Vers-
viers, Belgium) and the cells were collected
at 900g for 10 min. The cells were then layered
on a Percoll (Sigma Aldrich, St. Louis, MO)
gradient (density: 1.073 g/ml) and centrifuged
at 1,100g for 30 min, according to a previously
reported method (17). The cells in the inter-
phase were recuperated, washed twice with
PBS 1? (200g for 10 min) and seeded at a
density of 800.000/cm2in alfa-MEM (Cambrex
Bioscience) at 10% of fetal bovine serum (FBS)
in 75 cm2T-flasks (Greiner Bio-One GmbH,
Frickenhausen, Germany) and maintained at
378C with an atmosphere of 5% CO2. After
re-feed every 3–4 days.
In order to expand the isolated cells, the
adhered monolayer was detached with trypsin/
EDTA (Cambrex Bioscience) for 5 min at 378C,
after 15 days for the first passage and every
7 days for successive passages. During in vitro
passaging, the cells were seeded at a density of
8.000/cm2and expanded for several passages
until they no longer reached confluence.
Analysis of MSCs
At each passage the cells were counted and
analyzed for cellular growth, viability, and
immunophenotype analysis by cytofluorimetric
analysis. The evaluation of telomere length and
the karyotype analysis was also performed.
Cellular expansion growth rate was evalu-
ated by cell count in a Burker Chamber at each
passage and expressed in terms of population
doubling (PD) as performed in the studies
reported by Stenderup et al. .
Isolation of Mesenchymal Stem Cells745
Cytofluorimetric Analysis of MSCs
The identification of adherent cells was
performed by flow cytometry analysis. At each
passage 200–500 cells were stained for 20 min
with anti-CD45 fluoroisothyocyanate (FITC),
CD14 phycoerytrin (PE), CD90FITC, CD106-
PE, CD29FITC, CD44PE, CD105PE, CD166F-
ITC, and with 7-aminoactinomycin D (AAD)
(Becton Dickinson, San Jose, CA) for the vi-
ability. Labeled cells were thoroughly washed
with PBS 1? and were analyzed on a Epics XL
cytometer (Beckman Coulter, CA) with the XL2
software program. The percentage of positive
cells was calculated using the cells stained with
Ig FITC/PE as a negative control.
Evaluation of Telomere Length
In order to determine senescence during cul-
ture, telomere length was analyzed at each
passage by flow fluorescence in situ hybridiza-
tion (Flow-FISH) [Rufer et al., 1998]. Two
hundred thousand expanded
labeled with a telomere-specific conjugated
(C3TA2)3peptide nucleotide acid (PNA) probe
the same number were analyzed as a control
without a probe. The same number of hemo-
poietic stem cells (HSCs) was isolated with an
immunomagnetic system using CD34 micro-
beads conjugated antibody from BM at the
moment of collection and used as examples of
FACS Calibur cytometer (Beckon Dickinson).
At the beginning of each experiment, the
fluorescence signals from four different popula-
tions of FITC-labeled microbeads (Quantum-
TM-24 FITC Premix, Flow Cytometry Cor-
poration, IN) were acquired. The voltage and
amplification of the FL1 parameter were set in
molecular equivalents of soluble fluorochrome
(MESF)units per beadcorrespondedtochannel
numbers ranging from 10 to 15, 150 to 160, 450
to 460, 800 to 820, respectively, in the FL1
channel on a linear scale. The resulting calibra-
tion curve (y¼0.029x) was used to convert
telomere fluorescence into MESF units, to
compare the experiment results.
MSCs were cultivated overnight with fibro-
blast growth factor (FGF) (Sigma Aldrich),
Grand Island, NY) 100 ng/ml for 24 h before
harvesting with trypsin-EDTA. The cells were
then lysed with hypotonic KCL and fixed in a
solution of methanol/acetic acid (3:1). Fifty
metaphases were analyzed after GTG banding
using MackType software (Nikon Corporation,
Statistical analysis was performed using
Statistica StatSoft, version 6.0, software. Data
were assessed using Student’s t-test, used to
compare the means of the two groups of adults
and pediatric donors.
Seventeen BM samples were collected from
donors (6 male and 11 female) over 18 years of
age (range of age: 20–50 years) and 8 (4 male
and 4 female) with ages younger than 18 years
(range of age: 2–13 years). All donors had no
evidence of any concurrent illness and were not
receiving any medications that could affect
bone. The study was conducted according to
the Helsinki Declaration.
Isolation of MSCs
Adherent cells were observed in all samples
an adherent monolayer was achieved. BM cells
rapidly generated a confluent layer of cells
possessing an elongated, fibroblastic shape. No
morphological differences were observed on the
MSCs isolated from adult and pediatric donors,
but when early passage cells were compared
with late passage cells, MSCs showed a dif-
ferent morphology. The cells increased in size
and showed a polygonal morphology with
evident filaments in the cytoplasm especially
when isolated from the adult donors.
Analysis of MSCs
MSCs isolated from pediatric donors were
expanded for a median of 20 passages (range:
were expanded for a median of 18 passages
(range: 6–26 passages) before the growth
stopped. The cell growth of expanded in vitro
MSCs was strictly related to the donor’s age. In
fact, in vitro expanded MSCs isolated from
pediatric donors showed a median fold increase
of 3.1 (range: 1.6–4.6), 1.5 (range: 1.0–2.4), 1.3
746Mareschi et al.
(range: 0.2–1.9) at each passage after 5, 10, 15
passages, respectively, and reached a mean
number of cumulative PDs at the 10th passage
In the MSCs isolated from adult donors, the
median fold increase at each passage was of 1.8
(range: 0.9–3.9), 1.5 (range: 0.9–2.4), 1.1
(range: 0.2–1.5) after 5, 10, and 15 passages,
respectively, while the cumulative PD after
(A) and in adult (B) group are given in Figure 1.
the two groups showed a showed a significant
statistic difference (P<0.05).
A: Long-term growth curves each obtained from an individual pediatric donor (n¼8). B: Long-term growth
curves each obtained from an individual adult young donor (n¼17). C: Mean values of pediatric (*) and
adult donors (&). Each point represents mean?SD. *P<0.05. PD, population doubling; bp, base pair.
Growth kinetics of human MSCs culture showing cumulative PD as a function of time in culture.
Isolation of Mesenchymal Stem Cells747
TABLE IA. Median Values of the Antigens Expression (With Ranger Expressed in Percentage) of MSCs Isolated From Pediatric Donors
and Analyzed at Each Passage
3.66% (0.88–16.44) 2.20% (1.45–2.94)
8.37% (4.53–12.20) 10.67% (6.02–15.32) 71.75% (45.50–98.00) 97.85% (97.80–98.20) 97.95% (97.60–98.30)
6.65% (0.52–15.90) 8.42% (0.97–17.40)
8.21% (1.87–72.40) 82.75% (57.10–98.20) 98.75% (72.90–99.90) 96.25% (89.90–99.50)
63.40% (0.27–75.50) 17.70% (0.49–71.00) 87.30% (81.40–99.30) 91.35% (41.96–99.80) 96.20% (84.10–100.00)
4.42% (0.02–16.10) 4.08% (0.27–23.40)
36.90% (0.69–66.20) 36.30% (0.39–92.50) 85.20% (79.96–96.20) 90.85% (39.30–98.30) 83.75% (74.10–95.70)
4.75% (0.14–15.39) 1.70% (0.18–19.52)
27.30% (0.45–98.30) 29.00% (2.32–73.50) 98.65% (82.50–99.40) 97.50% (84.60–99.70) 92.65% (83.90–99.60)
1.01% (0.05–22.70) 8.47% (0.71–20.90)
20.72% (0.90–98.30) 13.80% (1.55–56.90) 84.80% (54.70–89.80) 93.75% (84.60–99.70) 81.40% (64.80–99.40)
3.50% (0.76–10.36) 3.40% (0.32–18.70)
32.50% (0.80–91.90) 31.30% (4.67–60.30) 89.80% (43.80–95.80) 96.60% (96.00–99.20) 98.00% (70.20–99.30)
3.17% (0.35–10.99) 2.54% (1.10–13.97)
27.81% (2.92–52.70) 23.86% (3.42–44.30) 92.70% (89.00–93.60) 91.95% (89.30–94.60) 92.80% (89.70–95.90)
4.23% (0.29–13.10) 4.22% (2.07–7.99)
9.72% (2.00–82.70) 84.20% (33.90–95.00) 98.85% (97.30–99.20) 97.30% (96.80–97.40)
Passage 10 4.32% (0.06–11.25) 4.50% (2.00–9.12)
99.00% (47.90–100.00) 20.60% (2.90–52.60) 32.30% (3.25–89.60) 80.50% (35.20–98.30) 99.20% (93.56–99.80) 98.70% (97.10–99.20)
TABLE IB. Median Values of the Antigens Expression (With Ranger Expressed in Percentage) of MSCs Isolated From Young Adult Donors
and Analyzed at Each Passage
12.67% (1.40–2.90) 95.85% (46.10–98.00) 95.40% (92.40–99.30)
67.95% (3.67–78.50) 93.00% (63.20–98.90)
94.60% (74.30–99.10) 97.70% (54.50–99.10)
21.30% (1.62–61.60) 88.30% (8.72–97.40)
19.00% (1.58–57.00) 91.20% (1.92–97.30)
11.70% (2.72–33.30) 77.50% (69.6–96.8)
85.30% (42.40–96.10) 93.85% (81.20–93.20)
11.94% (2.53–18.56) 75.80% (35.00–97.70)
25.33% (1.30–64.20) 77.35% (32.30–98.40)
96.50% (95.70–99.50) 98.30% (98.30–99.70)
20.43% (2.23–51.32) 86.00% (33.20–97.80)
84.40% (77.20–95.50) 96.00% (94.53–98.20)
29.80% (1.89–45.60) 78.30% (34.32–98.21)
86.70% (83.20–85.96) 97.60% (95.32–98.69)
6.26% (2.03–11.30) 76.90% (29.53–89.23)
0.42% (0.03–1.020) 86.90% (83.26–86.96) 94.50% (92.35–97.32)
7.27% (3.05–12.36) 78.63% (32.69–92.36)
Trypan blue staining analysis showed a
viability of between 98% and 100% in all
samples analyzed with no differences between
the two groups. The same results were con-
firmed after 7AAD staining in cytofluorimetric
Immunophenotype Analysis by
During the first 10 passages, the cells were
analyzed at each passage for the expression of:
CD45 and CD14, hemopoietic surface antigens;
CD90 (a membrane glycoprotein, also called
Thy-1), used as a stem cell marker; CD29, the b
subunit of the fibronectin receptor; CD44,
receptor-III of extracellular matrix; CD105 or
endoglin; CD166 and CD106, cell adhesion
Tables IA and IB show the median values of
the antigen expression (with ranges) of MSCs
isolated from pediatric and adult donors,
respectively, and analyzed at each passage.
At the first passage, MSCs isolated from
pediatric donors were CD45, CD14 negative
because antigen expression was less than 5%
(the median was 3.66% with a range of 0.88%–
respectively), while they showed a high exp-
ression of CD90 (median of 70.75%, range:
43.80%–97.70%), CD29 (median of 71.75%,
range: 45.50%–98.00%), CD44 (median of
97.85%, range: 97.80%–98.20%), and CD105
(median of 97.95%, range: 97.60%–98.30%)
molecules (median of 8.37% with a range
of 4.53%–12.20% and 10.67% with a range of
6.02%–15.32%, respectively). During the ex-
pansion time, the MSCs were negative for the
hemopoietic antigen while at each passage they
expressed high percentages of CD90, CD29,
antigen expression being over 80% (Fig. 2A).
expansion. Median values of immunophenotypic antigens in MSCs isolated from pediatric (A) and young
adults (B) during expansion from the 1st to the 10th passages are indicated. The percentage of positive cells
was calculated using the Ig FITC/PE stained cells as a negative control.
Modulation of median values of antigen expression analyzed at each culture passage during
Isolation of Mesenchymal Stem Cells749
At first passage, the MSCs isolated from
of hemopoietic cells (a median of 15.45% with a
range of 1.55%–29.50% of CD45 and 12.67%
with a range of 1.40%–32.90% of CD14 positive
cells) which decreased in the following pass-
ages. A high level of CD90, CD44 and CD105,
and CD29was expressed from these cells with a
median of 95.85% (range: 46.10%–98.00%),
95.40% (range: 92.40%–99.30%), and 96.10%
(range: 92.20%–98.50%) at the first passage
of expansion as shown in Figure 2B. The
expression of CD106 and CD166 antigens was
variable during the expansion in both groups.
Therewas no significantdifferencebetween the
modulation of antigen expression in the MSCs
isolated from the two groups.
In one only sample (MSC culture from an
adult donor) was the hemopoietic contamina-
tion persistent until the 10th passage and this
sample was excluded from the analysis of the
modulation of the antigen expression.
Evaluation of Cellular Senescence
Ten MSC samples (7 from pediatric and 3
from adult donors) were analyzed for cellular
senescence after 10 cumulative PDs in the
expansion. The mean telomere length at the
first PD showed no significant differences
donors with a mean of 10.11?0.5 kb or of
10.30?0.70 kb. After 10 PDs, the mean telo-
mere length was 8.36?0.75 kb and 9.2?1.4 kb
withamean shortening value of1.8?0.6 andof
1.1?0.8 (P¼0.3) in pediatric and adult donors,
In four samples (three pediatric donors and
passage obtained by Flow-FISH was compared
with the telomere length of CD34 positive cells
sorted at the moment of BM collection and
the telomere length of CD34 positive cells were
comparable with or slightly lower than the
MSCs at the start of the culture. In these sam-
ples, the median value of telomere length at the
1st passage was 10.22?0.43 kb against a
median value of CD34þ cells of 9.16?1.18 kb.
During MSC expansion, the telomere short-
ening was 1.8?0.17 kb (mean value) after
10 cumulative PDs. There was a significant
10 PDs in a group of three pediatric donors
(P¼0.024). The shortening trend was not
always linear and in one sample during the
expansion, the telomere length increased by
2.3 kb. The increase of the telomere length was
noted in one sample where the MSCs were
isolated and expanded from a 2-year-old child
and the karyotypic analysis of this was normal
at the 10th passage. Figure 3 shows the modu-
lation of telomere shorting, expressed in bp, in
relation with cumulative PD during the expan-
sion in pediatric (A) and adult (B) donors.
Five samples were analyzed at the 2nd, 5th,
and 10th passage and no chromosomal altera-
tion was noted. Two samples analyzed at
10th expansion passage did not show a suffi-
cient number of metaphases to perform the
On the basis of their capacity to adhere to
plastic, we isolated MSCs from BM of pediatric
and young donors using a Percoll gradient and
expanded them in alfa-MEMþ10% FBS with-
out adding growth factor. As we previously
reported [Lazarus et al., 2005], these multi-
potent cells had substantial proliferation and
expansion in culture and did not differentiate
spontaneously during culture expansion, but
did differentiate in adipocytes, chondrocytes,
and osteoblasts when they grew in lineage-
specific culture conditions.
anadherent layerinitially formed byindividual
cells, which rapidly reached confluence. We
observed an exponential growth of these cells
with 98% of viability at each passage and noted
that cell growth of expanded in vitro MSCs was
strictly related to the donor’s age. Indeed, the
MSCs isolated from pediatric donors reached a
cumulative PD almost twice as high as MSCs
isolated from young adult donors after 112 days
(10.2?1.9 vs. 5.5?3.7). In the successive
passages, MSCs isolated from adult donors
increased in size and showed a polygonal
morphology and the different morphology was
related to a decreasing proliferation capacity
(data not shown).
Our data are in accordance with a series of
between donor age and the number and the
proliferative capacity of MSCs isolated from
750 Mareschi et al.
young and old donors in short-term early
and Mitsui, 1976; Majors et al., 1997; D’Ippolito
et al., 1999; Nishida et al., 1999; Stenderup
et al., 2003]. However, there are no studies on
the modulation of MSCs isolated from pediatric
and young adult donors.
Furthermore, we analyzed the modulation of
antigen expression in the MSCs isolated from
two groups until 10th passage (77 days). At the
first passage, MSCs showed a low contamina-
tion of hemopoietic cells which became insignif-
icant (<5%) in the following passages during
expansion. There was a high expression of
CD90, CD29, CD44, and CD105 at the start
ofMSC culture and ateachpassagebecause the
antigen expression median was always more
adhesion molecules, showed a variable and
moderate expression during the expansion. As
far as antigens are concerned there was no
significant difference between the modulation
ofantigenexpressionin theMSCsisolated from
the two groups. It was also noted that hemo-
poietic contamination was more frequent in
adult donor MSCs. Pediatric donor MSCs
Telomere length expressed by bp in adult MSCs (n¼3). PD, population doubling; bp, base pair.
Isolation of Mesenchymal Stem Cells751
probably have a higher proliferative advantage
than adult donor MSCs.
Extensive proliferation, however, can induce
cells senescence, which was analyzed by telo-
mere length measurement by Flow-Fish. This
cytofluorimetric method was validated and
compared with the molecular method by Rufer
et al.  and has the additional advantage
that the analysis can also be made on a small
quantity of cells. Telomere length between the
two groups was similar at the start and its
shortening during expansion was not signifi-
cantly different, data which differed from our
expectations and from Baxter et al. .
They, in fact found that mean telomere restric-
tion fragment (mTRF) in pediatric MSCs was
significantly longer than adult MSCs. Adult
donor age, Baxter’s study, was between 59 and
75 years, with an age range much higher than
our donors. We analyzed telomere shortening
from cells at growth arrest after about 10
cumulativePDs,which wastheminimal expan-
sion of MSCs used for transplantation in
Osteogenesis Imperfecta [Horwitz et al., 1999]
and there was significant difference between
the two groups (1.8?0.6 and 1.1?0.8 in
et al. , an equal number of PDs in vitro
causes equivalent telomere erosion regardless
of donor age. In one sample of the pediatric
group, during expansion, the telomere length
increased by2.3kb.Onelimit ofourstudyisthe
low number of analyzed samples. However,
comparing the telomere shorting with cumula-
tive PD, we found that the average total loss at
each cellular division was less than 200 bp in
each group and these data can be correlated
with a physiological phenomenon, as reported
by different groups [Harley et al., 1990; Allsopp
et al., 1992] and, more recently, by Gammaitoni
(but not karyotypic alteration) observed in only
one sample, where the MSCs were isolated and
expanded from a 2-year-old child, might be due
to the immature state of these cells.
Recent studies [Serakinci et al., 2004; Rubio
et al., 2005] have show the human adult
mesenchymal stem cells (MSCs) can become
neoplastic cells after long-term in vitro culture.
Serakinci et al.  analyzed the expression
of the transformed phenotype during long-term
culture of three different cell lines derived from
MSCs transduced with the telomerase hTER
gene at various PD levels. The authors showed
that the MSCs acquired gene aberration and
in immune-deficient mice, only after reaching
elevatedPD levels(256PD). On the other hand,
Rubio’s more alarming study reported a spon-
taneous transformation of MSCs which, after
spontaneously bypassing the senescence phase,
accelerated the cell cycle rate compared with
pre-senescence MSCs. The senescence phase
was evident after about 2 months from the
isolation and the senescence duration varied
from 1 to 8 weeks. Moreover, the authors also
morphologically distinguished the cells into
a senescence phase and an in cell crisis.
long-term ex vivo expanded cells in clinical
However, our data excluded chromosomic
alterations, early cellular senescence, no mod-
ification of the immunophenotype during the
level. All these data suggest that MSCs can be
isolated and expanded from most healthy
donors irrespective of age, since the donor’s
age is only correlated to a defect of cellular
growthkinetics,maintaining theother peculiar
The variability of results depends on the
all, on the heterogeneous nature of the MSC
istics might depend on the donor to donor
They observed that growth properties and
functional capabilities of the MSCs might be
different, not only on the basis of the donor
nature, but also on samples obtained at differ-
ent times from the same donor.
There might be numerous clinical appli-
cations of MSCs. In particular, promising
results have been obtained using human MSCs
in clinical trials for Osteogenis Imperfecta
[Horwitz et al., 1999, 2001, 2002], metachro-
matic leukodystrophy, and Hurley syndrome
[Koc et al., 2002]. In addition, for their immu-
nosuppressive proprieties and capacity to sup-
port hemopoiesis, MSCs have been used to
reduce acute and chronic graft versus disease
and facilitate the hemopoietic engraftment
after BM transplantation [Koc et al., 2000;
tested in clinical trials for tissue regeneration
and engineering [Horwitz et al., 2002; Mangi
et al., 2003].
752 Mareschi et al.
It is now clear that MSCs might be a useful
multiple diseases. Our data regarding healthy
donor BM showed that MSCs isolated and
expanded from pediatric and adult young
donors did not present any fundamental dif-
ferences regarding cellular characterization,
have a faster growth rate than MSCs isolated
from adult donors. These results might reflect
the higher proliferative potential of pediatric
MSCs than adult MSCs and suggest an
improvement of a standard protocol of isolation
and ex vivo expansion for experimental and
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