Isolation and characterization of mesenchymal stem cells derived from bone

marrow of patients with Parkinson's disease

Author(s): Zhiqing Zhang, Xiaofang Wang, Suping Wang
Source: In Vitro Cellular & Developmental Biology - Animal, 44(5):169-177.
Published By: Society for In Vitro Biology
https://doi.org/10.1007/s11626-008-9093-1
URL: http://www.bioone.org/doi/full/10.1007/s11626-008-9093-1

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In Vitro Cell.Dev.Biol.—Animal (2008) 44:169–177
DOI 10.1007/s11626-008-9093-1
Isolation and characterization of mesenchymal stem cells
derived from bone marrow of patients
with Parkinson’s disease
Zhiqing Zhang & Xiaofang Wang & Suping Wang
Received: 10 December 2007 / Accepted: 29 February 2008 / Published online: 10 April 2008 / Editor: J. Denry Sato
# The Society for In Vitro Biology 2008
Abstract Mesenchymal stem cells (MSCs) are capable of
self-renewing and differentiating into multiple tissues; they
are expected to become a source of cells for regenerative
therapy. Compared to allogeneic MSCs, autologous MSCs
from patients needing cell-based therapy may be an ideal
alternative stem cell source. However, characterizations of
MSCs from a disease state remains extremely limited.
Therefore, we have isolated and characterized MSCs from
Parkinson’s disease (PD) patients and compared them with
MSCs derived from normal adult bone marrow. Our results
show that PD-derived MSCs are similar to normal MSCs in
phenotype, morphology, and multidifferentiation capacity.
Moreover, PD-derived MSCs are capable of differentiating
into neurons in a specific medium with up to 30% having
the characteristics of dopamine cells. At last, PD-derived
MSCs could inhibit T-lymphocyte proliferation induced by
mitogens. These findings indicate that MSCs derived from
PD patients’ bone marrow may be a promising cell type for
cellular therapy and somatic gene therapy applications.
Keywords Mesenchymal stem cells . Bone marrow .
Parkinson’s disease . Immunosuppression
Z. Zhang (*) : S. Wang
Department of Neurology, Dalian Central Hospital,
Dalian 116033, People’s Republic of China
e-mail: zhangzhiqing1972@yahoo.com.cn
X. Wang
Department of Hematology and Oncology,
the Oncology Hospital of Tianjin Medical University,
Tianjin, People’s Republic of China
Introduction
Bone marrow stroma contains multipotential mesenchymal
stem cells (MSCs) capable of supporting hematopoietic
stem cell growth, self-renewing, and differentiating into
various tissues of mesenchymal lineages, such as bone,
cartilage, tendon, muscle, and fat under specific conditions
(Horwitz et al. 1999; Makino et al. 1999; Pittenger et al.
1999; Jiang et al. 2002; Zhao et al. 2002; Kasten et al.
2006). Moreover, MSCs can be obtained from adult
organisms and can be expanded more than 1 billion fold
in ideal culture without the loss of their characteristics of
stem cells. Due to these abilities, MSCs are of great
potential in the context of tissue engineering and cell-based
therapy (Minguell et al. 2000; Le Blanc et al. 2004).
Previous studies have indicated that both mice and
human MSCs could be induced to differentiate into neurons
in vitro by β-mercaptoethanol or dimethylsulfoxide, and
butylated hydroxyanisol or in vivo by engraftment (Mezey
et al. 2000; Sanchez-Ramos et al. 2000; Woodbury et al.
2000; Brazelton et al. 2000; Zhang et al. 2006). Kopen et al.
(1999) injected MSCs into the lateral ventricle of neonatal
mice and found that transplanted MSCs could differentiate
into neurons when exposed to the brain microenvironment,
suggesting that MSCs are potentially useful as vectors for
treating a variety of central nervous system disorders.
Recently, studies demonstrated that MSCs could be induced
into tyrosine hydroxylase-positive and dopamine-producing
neuronal cells in vitro. Transplantation of these induced
cells showed improvement in apomorphine-induced rota-
tional behavior and adjusting step and paw-reaching tests
following intrastriatal implantation in a 6-hydroxy dopa-
mine rat model of Parkinson’s disease (PD; Dezawa et al.
2004). These findings demonstrated that the ability to
170 ZHANG ET AL.
transdifferentiate MSCs into special neurons could have a
substantial potential for treating neurodegenerative disorders.
Compare to allogeneic MSCs, autologous MSCs are
more suitable for clinical applications, especially for those
needing cell-based therapy. However, previous studies
only demonstrated the characteristics of MSCs derived
from health donors, and the definitive characteristics of
MSCs derived from disease states remain extremely
limited. In this study, we have made an attempt to isolate
and culture autologous MSCs from PD patients’ bone
marrow and examine their biology characteristics. Our
data showed that autologous MSCs derived from PD
patients display a fibroblast-like morphology, express
mesenchymal phenotype, and are able to differentiate into
adipocytes, osteoblasts, and neural cells in vitro. More-
over, PD-derived MSCs are capable of differentiating into
neurons under specific conditions with up to 30% having
the characteristics of dopamine cells. At last, PD-derived
MSCs possessed hematopoietic supportive ability and
could inhibit T lymphocyte proliferation induced by
mitogens.
Materials and Methods
Isolation of MSCs from PD patients. Eighteen patients with
PD (11 men, 7 women) and eight (five men, three women)
normal healthy volunteers were investigated in this study.
The mean age of the PD patients was 60.7 years (range 48–
69), and the mean age of the controls was 58.4 years (range
52–64). None of the patients had received any antiparkin-
sonian medication. None of the control subjects had a
history of neurologic or psychiatric diseases. After receiv-
ing informed consent from the patients according to the
academic guidelines on the use of human subjects in
research, human bone marrow was obtained from PD
patients and healthy donors under the protocol approved
by the institutional review board of the Dalian Central
Hospital. Bone marrow samples were taken from the iliac
crest of normal adults or PD patients. Mononuclear cells
were separated by a Ficoll–Paque gradient centrifugation
(specific gravity 1.077 g/mL; Sigma Diagnostics, St Louis,
MO) and cultured in expansion medium at 37° C with 5%
CO2 in a fully humidified atmosphere. The expansion
medium contained 60% Dulbecco’s modified Eagle’s
medium (DMEM)/F-12 (Gibco Life Technologies, Paisley,
UK), 40% MCDB-201 (Sigma), 10% fetal calf serum (FCS;
Gibco), 100 U/mL penicillin, and 1,000 U/mL streptomycin
(Gibco). After culture for 2 d, the culture medium was
replaced, and nonadherent cells were removed. Once cells
were more than 80% confluent, they were detached with
0.25% trypsin–ethylenediamine tetraacetic acid (EDTA;
Sigma), and then CD14-positive cells were depleted using
CD14 micromagnetic beads (Miltenyi Biotec, Auburn,
CA), and CD14-negative cells were replated.
Quantification of DNA content and cell growth patterns.
During the log phase of growth, the expanded MSCs were
trypsinized, permeabilized with 70% ethanol for 20 min at
4° C, washed with phosphate-buffered saline (PBS), then
treated with 40 μg/mL RNAse A for 20 min at 37° C and in
10 μg/mL propidium iodine for 5 min at room temperature.
Deoxyribonucleic acid (DNA) content was analyzed in
FACScan flow cytometer (Becton Dickinson, Franklin
Lakes, NJ) using the Cellquest software. To study the
growth pattern of the expanded MSCs, cells were seeded at
a concentration of 2×103 cells per well in a 24-well plate.
The adherent cells were trypsinized and counted by the
0.4% trypan blue exclusion method (dead cells would be
stained by trypan blue and thus excluded) at different time
points. Moreover, doubling time of the MSCs for passages
3–15 was determined by seeding 1×105 cells into 25-cm2
T-flasks, which were fed with expanded medium every 2 d
and then trypsinized and counted by the 0.4% trypan blue
exclusion method after 4 d. Mean doubling time was
calculated from days 0 to 4 for four different origin MSCs.
FACS analysis. For immunophenotype analysis, MSCs
were washed with PBS containing 0.5% bovine serum
albumin (Sigma) and incubated with primary antibodies
(10–20 ng/mL) for 30 min at 4° C. For intracellular
antigens detection, cells were fixed in 2% paraformalde-
hyde for 15 min at 4° C and permeabilized with 0.1%
saponin (Sigma) for 1 h at room temperature. Primary
antibodies included mAb against CD11a, CD11b, CD14,
CD29, CD31, CD34, CD44, CD45, CD105, CD106, GlyA,
and HLA-DR (BD Biosciences Pharmingen, San Diego,
CA). We used same-species, same-isotype irrelevant anti-
body as the negative control. Then, cells were washed and
incubated with fluorescein isothiocyanate (FITC)-conjugat-
ed secondary antibodies for 30 min at 4° C. Cell analysis
was performed with a FACSCalibur system using CellQuest
software. Each measurement contained 10,000 cells.
Karyotyping analysis. MSCs derived from PD or normal
adults were analysis for karyotype. All cells in the log
phase of growth were processed by 0.1 μM cochineals for
6 h at 37° C and then digested with 0.25% trypsin–EDTA
and subjected to a 15 min colcemid (Sigma) incubation
followed by lysis with 0.075 mM KCl and fixation in
methanol/acetic acid (3:1). Metaphases were analyzed after
QFQ or GTG banding.
Transmission electron microscope analysis. Once expand-
ed MSCs were more than 90% confluent, they were
detached with 0.25% trypsin–0.04%EDTA and centrifuged
 FUNCTION OF AUTOLOGOUS MESENCHYMAL STEM CELL
at 1,200 rpm for 5 min, and then the cells blocks were fixed
in 6% glutaraldehyde and 1% OsO4. After dehydration in
acetone, cells blocks were embedded in GP-812 resin and
cut into ultrathin sections. Sections were double-stained
with uranyl acetate and lead citrate before observation
under an electron microscope.
Multilineage differentiation of MSCs. For osteoblasts dif-
ferentiations, MSCs were cultured at 2–3×104/cm2 in
DMEM with 10 mM β-glycerophosphate, 10−7 M dexa-
methasone, and 0.2 mM ascorbic acid. After 21 d,
differentiation to osteoblasts was shown by Von Kossa
stain for calcium phosphate and reverse transcriptase (RT)-
polymerase chain reaction (PCR) for osteopontin; For
adipocyte differentiations, expanded clonal MSCs were
cultured at 2–3×104/cm2 in DMEM with 10% FCS, 10−6 M
dexamethasone, 50 μg/mL ascorbic acid, and 100 μg/mL 1-
methyl -3- isobutyl-xanthine (Sigma). After 14 d, oil-red
staining and RT-PCR for lipoprotein lipase showed adipo-
cyte differentiation; for chondrogenic differentiation, 1×106
MSCs were cultured in 1 mL serum-free expansion medium
with 100 ng/mL transforming growth factor 1, in the tip of
a 15-mL conical tube to allow aggregation of the cells in
the micromass suspension culture. The development of
chondroblasts was followed by staining the micromasses
with toluidine blue, which stains the proteoglycan extracel-
lular matrix (ECM), and with antibodies against type II
collagen. For neural differentiation, MSCs were cultured in
a 24-well plate at 2×104 cells per well in α-modified
minimum essential medium (αMEM) with 10% FCS,
10 ng/mL basic fibroblast growth factor (bFGF; Peprotech,
Rocky Hill, NJ), 50 ng/mL brain-derived neurotrophic
factor (Peprotech), and 50 ng/mL of Nerve growth factor
(Takara Shuzo, Tokyo, Japan). The differentiated cells were
observed by phase-contrast microscopy and detected by
fluorescence-activated cell sorting (FACS), immunocyto-
chemistry, and Western blotting for neuron-specific markers
microtubule-associated protein 2 (MAP-2) and neurofila-
ment (NF), astrocyto-specific marker, glial fibrillary acidic
protein (GFAP), and oligodendrocytic-specific marker
galactocerebroside (GalC; Sanchez-Ramos et al. 2000).
Dopaminergic neuron differentiation of MSCs. For dopa-
minergic neuron differentiation, MSCs were cultured in a
24-well plate at 2×104cells per well in αMEM with 10%
FCS, 10 ng/mL bFGF (Peprotech), and 50 ng/mL of GDNF
(Peprotech). The differentiated cells were examined by
FACS, immunofluorescence, RT-PCR, and Western blotting
for dopaminergic neuron-specific marker tyrosine hydrox-
ylase (TH) and transcription factors Nurr-1, Lmx1b, and
Ptx3 (Sakurada et al. 1999; Dezawa et al. 2004). Further-
more, differentiated cells were examined by dopamine
release assay as described previously (Dezawa et al.
171
2004). Cells were washed twice with a low K+ solution
and incubated in the low K+ solution for 2 min, and then
the medium was replaced with a high K+ solution for 5 min.
Concentration of dopamine was determined by high-
performance liquid chromatography (HPLC) using a re-
verse-phase column and an electrochemical detector system
(Eicom, Kyoto, Japan; Dezawa et al. 2004).
Mixed leukocyte reaction. MSCs were irradiated (30 Gy)
and plated in triplicates onto 96-well plates at 1×104/mL in
100 μL complete media. Allogeneic CD2+ T cells purified
from peripheral blood mononuclear cells by using the
MACS CD4 isolation kit, resuspended at 1×105/mL, were
added to wells containing or lacking MSCs in the presence
of the mitogen phytohemagglutinin (PHA; 2.5 μg/mL). The
MSCs to CD4+ T cells ratio was 1:10. Cocultures without
PHA were used as controls. The culture was continued, and
3
H-thymidine (1 μCi [0.037 MBq]) was added 18 h before
the end of the 120-h culture. The T cell proliferation was
represented as the incorporated radioactivity in counts per
minute (cpm) and shown as means±SDs of triplicate
values.
Reverse transcriptase polymerase chain reaction. RT-PCR
for lipoprotein lipase, osteopontin, transcription factors
Nurr-1, Lmx1b, Ptx3, and β2-microglobulin was per-
formed. Total ribonucleic acid (RNA) was prepared with
the use of Trizol reagents in accordance with the protocol
recommended by the manufacturer and visualized by means
of UV spectrophotometry. We synthesized first-strand
complementary DNA (cDNA) from 4 μg of total RNA
primed with oligo-dT primer using the First-Strand cDNA
Syn-thesis Kit (Gibco). We amplified 1/50 of the RT
mixture with the use of the GeneAmp PCR System 2400
thermal cycler (Perkin-Elmer, Boston, MA) in a total
reaction volume of 20 μL containing 10 pmol of each
primer, 200 μmol/L of deoxyribinucleoside triphosphates,
and 2 U of Taq polymerase (Gibco). Primer sequences and
precise conditions are described elsewhere (Seta et al. 1999;
Zheng et al. 2000; Fang et al. 2005). The reaction products
were resolved by electrophoresis on a 1.5% agarose gel and
visualized with ethidium bromide.
Immunofluorescence and Western blotting. For immunoflu-
orescence, neural-like cells were fixed with 4% parafor-
maldehyde at room temperature, permeabilized them with
the use of 0.1% Triton X-100 in PBS for 10 min, and
treated them with 5% FCS in PBS for 30 min at room
temperature. Cells then were incubated for 1 h with diluted
primary antibodies at room temperature. Mouse anti-human
GFAP (BD Pharmingen; 1:1,000 dilution), GalC (Serotec,
Raleigh, NC; 1:500 dilution), and NF (Santa Cruz Biotech-
nology, Santa Cruz, CA; 1:100 dilution) were used. After
172 ZHANG ET AL.

incubated with these antibodies for 1 h at room tempera-

ture, samples were washed three times with PBS and then
incubated for 30 min at room temperature with anti-mouse
IgG/FITC (Sigma). Next, the slides were scrutinized, field
by field, with the use of fluorescence microscopy (Olympus;
Olympus Optical, Tokyo, Japan).
For Western blotting, equivalent amount of protein
lysates, obtained from MSCs or differentiated progeny,
were loaded per lane. After sodium dodecyl sulfate
polyacrylamide gel electrophoresis, proteins were electro-
phoretically transferred onto nitrocellulose membrane
(Amersham Pharmacia Biotech; Uppsala, Sweden). After
blocking, blots were incubated with mouse polyclonal
antibodies against GalC (Serotec; 1:500 dilution), GFAP
(BD Pharmingen; 1:1,000 dilution), MAP-2 (Dako, Carpin-
teria, CA; 1:100 dilution), NF (Santa Cruz Biotechnology;
1:100 dilution), and TH (1:200 dilution) for one night at 4° C
temperature. Expression of the β-actin was used as an
internal control. Immunodetection using the enhanced
chemiluminescence method (ECL kit; Amersham, Piscat-
away, NJ) was performed according to the manufacturer’s
instructions. Figure 1. Morphology and growth characteristic of PD MSCs. A. PD
MSCs were cultured at 2×103 cells per well in a 24-well plate. The
number of viable cells was measured by counting trypan blue-negative
cells. The population doubling time was about 50 h. B. Doubling time
of MSCs derived from normal and PD MSCs with successive passage.
Result Results are expressed as mean±SEM. Normal adult, diamonds, n=4;
PD, squares, n=4.
Biological characteristics of undifferentiated PD MSCs. In
this study, MSCs were obtained from the bone marrow of MSCs derived from PD and normal adults showed a normal
PD patients (n=18). We compare their biological character- karyotype.
istics with MSCs derived from bone marrow of normal
adults (n=8). The biological characteristics of MSCs were Ultrastructure of undifferentiated PD MSCs. Under trans-
investigated and compared over a 20-wk period. Our results mission electron, the nucleus of the MSCs contains one or
show that MSCs derived from PD and normal patients two small nucleoli surrounded by nucleolar-associated
displayed fibroblast-like morphology under phase-contrast chromatin. The cytoplasm contains a relatively small
microscopy. Two weeks later, almost all obtained MSCs (16 quantity of organelles, such as the endoplasmic reticulum,
of 18 for PD and eight of eight for normal adult) formed ribosomes, mitochondria, and Golgi complex. There are no
colony-forming unit fibroblast colonies (CFU-F). The granules in the cytoplasm. Moreover, normal and PD MSCs
frequency of CFU-F was similar among MSCs derived have a similar ultrastructure.
from PD and normal patients (4±1.89 and 5±2.14 CFU-F
per 106 bone marrow mononuclear cells for PD and normal Multiple differentiation of PD MSCs. To induce adipocyte
MSCs, respectively). A typical growth curve is shown in differentiation, there were lipid-laden cells that appeared
Fig. 1A. The doubling time of PD and normal-origin MSCs after 5 d. After 2 wk, more than 60% of MSCs
was similar (52.3±6.5 h for PD, 51.9±10.3 h for normal differentiated into adipocytes that stained with Oil-red O
adult, respectively). The doubling time of MSCs remained (Fig. 2A, C). Using RT-PCR, we also found that these cells
unchanged during culture expand for at least 20 passages expressed lipoprotein lipase, an adipocyte special gene
(Fig. 1B). On average, 91.34% cells were at G0/G1, 2.71% (Fig. 2E). At exposure of MSCs to the osteogenic medium,
at G2/M, and 5.95% at S phases of the cell cycle. The the cells formed aggregates, and there were calcium deposit
immunophenotype of PD MSCs was analyzed by FACS. appears after 3 wk. To confirm osteogenic differentiation,
PD MSCs expressed CD106, CD105, CD29, and CD44; Von Kossa staining for ECM with one osteogenic-specific
they did not express CD11a, CD11b, CD14, CD31, CD34, marker was performed, and we identified that calcification
CD45, GlyA, and HLA-DR (Table 1). This phenotype of the ECM was observed in induced MSCs (Fig. 2B, D).
remained unchanged for more than 20 passages. Moreover, Moreover, RT-PCR analysis also showed another osteogenic-
 FUNCTION OF AUTOLOGOUS MESENCHYMAL STEM CELL 173

Table 1. Immunophenotypes of cultured MSCs data showed that induced cells released 2.14 pmol/106 cells
Antigen PD MSCs (%) Normal adult MSCs (%) of dopamine to the culture media in response to high K+-
depolarizing stimuli, whereas dopamine release from
CD11a −0.8 (0.3–1.6) −0.9 (0.3–1.8) undifferentiated MSCs was under the detection level.
CD11b −1.2 (0.5–1.4) −1.1 (0.4–1.5)
CD14 −0.9 (0.4–1.5) −0.8 (0.7–1.2)
Inhibition of T lymphocyte proliferation. Previous reports
CD29 +99.7 (98.2–100) +98.1 (97.3–99.7)
reported that MSCs are capable of suppressing the
CD31 −0.7(0.3–1.7) −1.7 (0.8–2.5)
CD34 −1.3 (0.3–2.1) −0.7 (0.2–0.9) proliferation of T lymphocytes stimulated in a mixed-
CD44 +97.3 (96.2–98.4) +94.8 (93.2–99.4) lymphocyte culture. Therefore, the effect of MSCs on T cell
CD45 −1.0 (0.8–1.4) −0.4 (0.1–0.7) proliferation was evaluated by mixed-leukocyte reactions in
CD105 +96.4 (95.2–98.7) +95.8 (94.7–99.1) the presence of PHA. Our data showed that T lymphocyte
CD106 +94.9 (93.2–99.8) +97.2 (96.6–98.2) proliferation induced by PHA was markedly reduced in the
HLA-DR −1.2 (0.7–2.2) −0.9 (0.7–1.8) presence of PD MSCs or normal adult MSCs. The addition
Glay A −1.4 (0.9–1.8) −0.7 (0.4–1.3)
of MSCs to the mixed T lymphocyte culture could reduce T
− indicated no cells were positively stained; + indicated cells were lymphocyte proliferation by more than 70% (P≤0.01). The
positively stained. ability of inhibition of PHA-stimulated T cell proliferation
was similar between PD and normal adult bone marrow
specific gene, osteopontin, in the cells after osteogenic MSCs (Fig. 4).
induction (Fig. 2E). To induce chondrogenic differentiation,
after 2 wk, small aggregates of cartilage formed in the
bottom of the tubes were stained positive with toluidine blue
(Fig. 2F, G). Moreover, expression of type II collagen was Discussion
detected from day 5, and type II collagen staining could be
detected throughout the micromass (Fig. 2H). Exposure of PD is a common neurodegenerative disorder of the nerve
MSCs to the induced medium resulted in the expected system. The critical pathological change underlying the
changes in morphology as responsive cells assumed forms condition is the loss of the dopaminergic nigrostriatal
typical of nerve-like cells after 7 d. After 14 d, about 40% of pathway with the formation of α-synuclein-positive Lewy
the induced cells exhibited typical morphology of a nerve- bodies. Tremor and slow movements develop only after
like cell. Induced MSCs were assessed for the expression of about 70% of vulnerable dopaminergic neurons in the
neuronal-specific markers (NF) and MAP-2, in addition to substantia nigra has already died (Quinn 1997; Conway and
GFAP and GalC, as markers of astrocytes and oligoden- Harper 1998; Dauer and Przedborski 2003). Current
drocytes, respectively. FACS showed that 40% of induced therapeutic strategies for PD focus primarily on reducing
cells expressed NF. Immunofluorescence and Western the severity of its symptoms using dopaminergic drugs.
blotting confirmed the expression of NF and MAP-2 in Unfortunately, with time, these therapies fail and produce
induced cells. In contrast to NF or MAP-2, no expression of their own unique side effect profile, and this, coupled with
GalC or GFAP was noted, indicating that MSCs cells did not the more diffuse pathological and clinical findings in
differentiate into oligodendrocytes or astrocytes. (Fig. 2I, J) advancing disease, has led to a search for more effective
therapies (Dunnett and Bjorklund 1999; Jankovic 2002;
Dopaminergic neuron differentiation. After cultured in a Ahlskog 2003). More recently, cell-based therapy has
medium containing GDNF for 2 wk, more than 40% of emerged as a promising approach for restoration of function
MSCs differentiated into neuron-like cells that were in neurodegenerative diseases, in particular PD and Hun-
positive for NF. We performed immunocytochemical tington’s disease (Kawasaki et al. 2000; Perrier et al. 2004).
examination of transmitters and related proteins in those Previous studies demonstrated that embryonic stem cells
cells and found that induced cells were immunopositive for (ESCs), obtained from the early stages of embryonic
TH, one of the dopaminergic neuron-specific markers development, and neural stem cells (NSCs), obtained from
(Fig. 3A), and FACS showed that about 30% cells the developing brain, have been proposed as candidates
expressed TH. Western blot analysis further confirmed for transplantation therapy in neurodegenerative diseases
these results (Fig. 3B). Moreover, induced cells also express (Barberi et al. 2003; Park et al. 2004). However, the
the transcription factors of Nurr-1, Lmx1b, and Ptx3, which practical use of ESCs or NSCs was limited due to potential
are the transcription factors that have roles in the problems of cell regulation and ethical considerations.
differentiation of midbrain precursors into dopaminergic Recently, several studies demonstrated that MSCs were of
neurons (Quinn 1997; Fig. 3C). The amount of dopamine great therapeutic potential against neurological diseases
released upon depolarization was measured by HPLC. Our because of reasons as follows: Firstly, they can be readily
174 ZHANG ET AL.

Figure 2. MSCs differentiate into adipocyte, osteoblast, chondroblast, control. PD MSCs (F) and normal-derived
Figure 2. MSCs differentiate into adipocyte, osteoblast, chondroblast, and neuroectoderm in vitro. PD MSCs (A)MSCs (G) were
and normal bonecultured for
marrow-
and neuroectoderm in vitro. PD MSCs (A) and normal bone marrow-
derived MSCs (C) were cultured for 5 d in an induced medium; lipid-laden 2cells
wk appear
in the induced medium
in adipogenic and stained
medium, with adipocyte
and then, toluidine blue to identify
differentiation
derived MSCs (C) were cultured for 5 d in an induced medium; lipid-laden proteoglycan ECM. Moreover, the for
presence
was shown by Oil-red O staining. PD MSCs (B) and normal bone marrow-derived MSCs (D) were cultured 3 wk inofantype II collagen
osteogenic was
medium,
cells appear in adipogenic medium, and then, adipocyte differentiation was confirmed by Western blotfor
(H;lipoprotein
lane 1, induced PDosteopontin
MSCs; lane 2,
and osteoblast differentiation was indicated by Von Kossa staining for calcium phosphate. (E) RT-PCR lipase and gene.
shown by Oil-red O staining. PD MSCs (B) and normal bone marrow- uninduced PD MSCs; lane
The expression of lipoprotein lipase was detected in induced adipocytes, and the osteocalcin gene was3, detected
induced in normal-derived MSCs; (lane
induced osteoblasts lane 3,
4,
derived MSCs (D) were cultured for 3 wk in an osteogenic medium, and uninduced normal-derived MSCs; bottom, β-actin).
induced normal derived MSCs; lane 4, induced PD MSCs), and there was no expression in uninduced cells (lane 1, uninduced normal derived I After 7 d cultured in
osteoblast differentiation was indicated by Von Kossa staining for calcium
MSCs; lane 2, uninduced PD MSCs). β2-Microglobulin was performed asthe neurogenic
a control. medium,
PD MSCs (F)induced cells were stained
and normal-derived MSCspositively
(G) werefor NF. J
cultured
phosphate. (E) RT-PCR for lipoprotein lipase and osteopontin gene. The Neuronal differentiation was detected by Western blotting with monoclo-
for 2 wk in the induced medium and stained with toluidine blue to identify proteoglycan ECM. Moreover, the presence of type II collagen was
expression of lipoprotein lipase was detected in induced adipocytes, and nalPD
antibodies against NF, MAP2, GFAP, and GalC
confirmed by Western blot (H; lane 1, induced PD MSCs; lane 2, uninduced MSCs; lane 3, induced normal-derived MSCs;(lane lane1,4,induced
uninducedPD
the osteocalcin gene was detected in induced osteoblasts (lane 3, induced MSCs; lane 2, uninduced PD MSCs; lane 3, induced normal-derived
normal-derived MSCs; bottom, β-actin). I After 7 d cultured in the neurogenic medium, induced cells were stained positively for NF. J Neuronal
normal derived MSCs; lane 4, induced PD MSCs), and there was no
differentiation was detected by Western blotting with monoclonal antibodies MSCs; laneNF,
against 4, MAP2,
uninduced
GFAP, normal-derived
and GalC (lane MSCs; bottom,PDβ-actin).
1, induced MSCs;
expression in uninduced cells (lane 1, uninduced normal derived MSCs; Original magnifications: A, B, C, D, F, G, and I: 100×. Representative
lane 2, uninduced PD MSCs; lane 3, induced normal-derived MSCs; lane 4, uninduced normal-derived MSCs; bottom, β-actin). Original
lane 2, uninduced PD MSCs). β2-Microglobulin was performed as a example of six experiments.
 FUNCTION OF AUTOLOGOUS MESENCHYMAL STEM CELL 175

Figure
Figure 3.3. PD
PD MSCs
MSCs differentiate
differentiateinto
intodopaminergic
dopaminergicneuron
neurondifferentiation.
differen- A. Afterderived
normal cultured in medium
MSCs; bottom,containing
β-actin).GDNF forcultured
C. After 2 wk, induced cells
in medium
were stained
tiation. positively
A. After for TH.
cultured B. Dopaminergic
in medium neuron
containing GDNF differentiation
for 2 wk, wascontaining
detected by
GDNFWestern
for 2blotting with monoclonal
wk, induced antibodies
cells also express against TH.
the transcription
(lane 1, cells
induced uninduced PD MSCs;
were stained lane 2,
positively forinduced PD MSCs; lane
TH. B. Dopaminergic 3, uninduced
neuron factorsnormal derived
of Nurr-1, Lmx1b,MSCs;and lane
Ptx3 4, induced
(lane normal derived
1, uninduced MSCs;
normal derived
bottom, β-actin).
differentiation wasC. After cultured
detected in medium
by Western containing
blotting with GDNF for 2 wk,MSCs;
monoclonal inducedlane
cells2,also express PD
uninduced the MSCs;
transcription
lane 3,factors of Nurr-1,
induced Lmx1b,
normal-derived
and Ptx3 (lane
antibodies 1, uninduced
against normal
TH. (lane 1, derived
uninduced PDMSCs;
MSCs;lane
lane2,2,uninduced
induced PD MSCs;
MSCs; lane
lane 3, induced
4, induced PD normal-derived
MSCs; bottom, β MSCs; lane 4, induced PD
2-microglobulin). Original
MSCs;
PD MSCs; laneβ3,2-microglobulin).
bottom, uninduced normal Original magnification:
derived MSCs; lane A: 100×. Representative
4, induced example
magnification: of sixRepresentative
A: 100×. experiments. example of six experiments.

obtained through a well-established clinical procedure; last, we test the differentiation ability of different passage
secondly, they are easy to isolate and expand for auto- PD MSCs. They had the ability to differentiate into, at least,
transplantation with no risk of rejection; lastly, they are adipocytes, osteoblasts, chondroblasts, and neural cells in
capable of self-renewal and multiple differentiations (Deans vitro, and the differentiation ability of the fifth, tenth, and
and Moseley 2000; Koc and Lazarus 2001; Reyes et al. 15th passage PD MSCs had no difference. Previous studies
2001). had shown that stem cells possessed the capacities of self-
Although both allogeneic and autologous MSCs possess renewal and multidifferentiation, so our results demonstrate
the clinical therapeutic potential, allogeneic MSCs must that PD MSCs meet the criteria for stem cells.
face limitations inherent, such as histocompatibility, inad-
equate tissue supply, therapeutic scale, and ethical con-
cerns. Therefore, autologous MSCs from patients needing
cell-based therapy may be an ideal alternative stem cell
source and are more suitable for clinical applications. In
this study, we investigated the characteristics of MSCs
derived from patients with PD and compared them with
MSCs derived from normal adult bone marrow. Our data
showed that PD MSCs displayed fibroblast-like morphol-
ogy and could withstand continuous passage for at least 20
times. The morphology and phenotype of MSCs remained
unchanged with cell passage, implying that we could obtain
sufficient cell counts for cell-based therapy. PD MSCs
expressed several cell surface antigens commonly found on
so-called MSCs, such as SH2 (CD105), CD44, CD29, and Figure 4. Inhibitory effect of PD MSCs on T-lymphocyte prolifera-
tion. Allogeneic CD2+ T cells were incubated for 5 d with MSCs
CD106. Furthermore, the flow cytometer showed that about
derived from PD or normal adults in the presence of the mitogen PHA.
91% MSCs were in the G0/G1 phase, indicating that the Data are expressed as mean±SD of triplicate values. Asterisk, P≤0.05.
most of MSCs were in the undifferentiated early state. At Representative example of four experiments.
176 ZHANG ET AL.
The multidifferentiation capacity of MSCs has stimulat-
ed research aimed at the selective generation of specific
cells types for regeneration medicine. Previous studies had
shown that the coculture of human ESCs on stromal feeder
cells yields highly efficient differentiation into dopamine
neuron (Kawasaki et al. 2000; Lee et al. 2000). More
recently, Dezawa et al. (2004) reported that both rat and
human MSCs have the capability under specific conditions
of differentiating into dopamine neurons and that induced
cells may have properties useful in the treatment of PD.
Although dopamine neurons can be obtained form ESCs or
allogeneic MSCs, the inherent defects limit the application
of ESCs or allogeneic MSCs. Compared to ESCs or
allogeneic MSCs, autologous MSCs from patients needing
cell-based therapy may be an ideal alternative stem cell
source and are more suitable for clinical applications.
Similar to previous studies, our experiments had also
demonstrated that autologous MSCs derived from PD
patients could selectively differentiate into dopaminergic
neurons under an induced medium used before. We found
that about 30% induced cells were immunopositive for TH,
the rate-limiting enzyme in the synthesis of dopamine. In
addition to TH expression, induced cells also express the
transcription factors of Nurr-1, Lmx1b, and Ptx3, which are
the transcription factors that have roles in the differentiation
of midbrain precursors into dopaminergic neurons (Sakurada
et al. 1999). Moreover, induced cells could produce
dopamine, suggesting the efficient derivation of dopamine
neurons from PD MSCs.
Previous reports reported that MSCs are capable of
suppressing the proliferation of T lymphocytes stimulated
in a mixed-lymphocyte culture or by incubation with
mitogens (Di Nicola et al. 2002; Krampera et al. 2003;
Tse et al. 2003). The immunosuppressive ability of MSCs
has a substantial impact in transplantation and may reduce
the allogeneic immune response. Batholomew et al. (2002)
demonstrated that the infusion of allogeneic MSCs into
baboons was well tolerated in most animals and prolongs
the survival of allogeneic skin graft. Although the immu-
nomodulatory effects of MSCs have important implications
in transplantation biology, we do not know whether the
immunosuppressive effect of MSCs was affected by disease
state. In this study, we demonstrated that PD MSCs are able
to inhibit the T cell proliferation stimulated in a mixed-
lymphocyte culture or by incubation with mitogens. The
immunosuppressive effect was similar between PD MSCs
and normal adult MSCs, suggesting that PD MSCs still
preserved the immunosuppressive effects.
In summary, we described for the first time the
biological characteristics of MSCs derived from PD
patients’ bone marrow. Our results showed that PD-derived
MSCs are similar to normal MSCs in phenotype, morphol-
ogy, and multidifferentiation capacity. Moreover, PD-
derived MSCs are capable of differentiating into neurons
in tissue culture with up to 30% having the characteristics
of dopamine cells. At last, PD-derived MSCs could inhibit
T lymphocyte proliferation induced by mitogens. There-
fore, the successful isolation of MSCs from PD patients’
bone marrow and the examination of their function and
natural characteristics may lead to new therapeutic
approaches for this disease.
Acknowledgment The authors thank all staff of department of
Neurology of Dalian People Hospital for the donation and collection
of bone marrow samples.
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