JCP07-0027.

R1(21251)
ORIGINAL ARTICLE
Journal of
1
Cellular
Potential Conversion of Adult
Clavicle-Derived Chondrocytes
Author Proof Physiology

Into Neural Lineage Cells In Vitro

A
HONG-YUN LI,1,2 AND XIN-FU ZHOU1*
1
Department of Human Physiology, School of Medicine, Flinders University of South Australia, Adelaide,
South Australia, Australia
2
Department of CNS Trauma & Rehabilitation, Research Institute of Surgery, Daping Hospital, Chongqing, P.R. China

Neural stem cells (NSC) can be isolated from a variety of adult tissues and become a valuable cell source for the repair of peripheral and
central nervous diseases. However, their origin and identity remain controversial because of possible de-differentiation/trans-
differentiation or contaminations by hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs). We hypothesize that the
commonly used NSC culture medium can induce committed cartilage chondrocytes to de-differentiate and/or trans-differentiate into
neural cell lineages. Using a biological isolation and purification method with explants culture, we here show that adult rat clavicle cartilage
chondrocytes migrate out from tissue blocks, form sphere-like structures, possess the capability of self-renewal, express nestin and
p75NTR, markers for neural crest progenitors, and differentiate into neurons, glia, and smooth muscle cells. Comparing with adult
cartilage, the spherical-forming neural crest cell-like cells downregulate the chondrocytic marker genes, including collagen II, collagen X,
and sox9, as well as neural-lineage repressors/silencers REST and coREST, but upregulate a set of well-defined genes related to neural
crest cells and pro-neural potential. Nerve growth factor (NGF) and glial growth factor (GGF) increase glial and neuronal differentiation,
respectively. These results suggest that chondrocytes derived from adult clavicle cartilage can become neural crest stem-like cells and
acquire neuronal phenotypes in vitro. The possible de-differentiation/trans-differentiation mechanisms underlying the conversion were
discussed.
J. Cell. Physiol. 9999: 1–15, 2007. ß 2007 Wiley-Liss, Inc.

In the last 5 years, there is an explosion of new reports on
neural stem cells (NSC) isolated from a variety of sources such
as embryonic, fetal, and adult tissues. Neural stem/progenitors
cells (NSC) can be isolated from adult peripheral tissues
including bone morrow (D’Ippolito et al., 2004; Bonilla et al.,
2005; Bossolasco et al., 2005; Gregory et al., 2005; Hermann
et al., 2006), muscle (Romero-Ramos et al., 2002; Alessandri
et al., 2004; Vourc’h et al., 2004; Schultz and Lucas, 2006), skin
including dermis (Toma et al., 2001; Joannides et al., 2004;
Kawase et al., 2004; Wong et al., 2006) and hair follicle (Sieber-
Blum et al., 2004), gut (Kruger et al., 2002), pancreas (Eberhardt
et al., 2006), liver (Koenig et al., 2006), cornea (Yoshida et al.,
2006), sclera and the choroid (Arsenijevic et al., 2003), heart
(Tomita et al., 2005), as well as fat (Fraser et al., 2006). Although
many questions remain and require further investigation, the
consensus is that NSCs can be obtained from many adult tissues
and that these cells can be potentially used for neural repair or
neuroregeneration (Krabbe et al., 2005; Hermann et al., 2006).
However, the origin and identity of adult peripheral NSCs
remain controversial. The questions of whether they are
derived from stem cell niches/reserved stem cells, or from
contaminated precursors of other tissues, or resulted from de-
differentiation/trans-differentiation in vitro, needs further
investigation. These questions are valid, since the tissues or
organs for isolation of stem cells in most previous studies are of
heterogeneous nature and consist of many different cell types
such as blood, nerves, blood vessels, adipocytes or other
mesenchymal cells.
A plethora of recent evidence has indicated that de-
differentiation/trans-differentiation mechanism occurs in vitro.
For example, bone marrow mesenchymal stem cells (MSCs)
which are derived from mesoderm not only differentiate into
chondrocytes, osteoblast, and adipocytes but also differentiate
into hepatocytes of endoderm, and neurons and glial cells of
ectoderm (Woodbury et al., 2000; Krause et al., 2001; Jiang
et al., 2002; Woodbury et al., 2002; Hermann et al., 2004;
Bonilla et al., 2005). A direct trans-differentiation of bone
marrow-derived adult progenitor cells (MAPCs) into neural
progenitor cells was also reported (Chen et al., 2006).
In the present study, we hypothesize that a standard NSC
culture medium can induce committed or differentiated cells to
trans-differentiate into unrelated cell lineages in vitro.
Specifically, we hypothesize that differentiated chondrocytes
from neural crest origin can be converted into neural crest stem
cell-like cells and then differentiate into neurons and glia. To
test this idea, purified primary chondrocytes from neural crest
origin are necessary. Cartilage tissue is a unique tissue since it
only contains a single cell type, chondrocytes, and is full of
extracellular matrix (Goldring et al., 2006); more importantly,
cartilage is an avascular and aneural tissues (Magne et al., 2005;
Schipani, 2005; Hall, 2005a; Goldring et al., 2006) which do not
contain any endothelial cells, smooth muscle cells, Schwann
cells, fibroblast, and other hematopoietic cells. Thus, it is a
desirable tissue for explants culture without possible
contamination of other lineage cells. In the present study we
chose the clavicle bone to isolate cartilage as it is the only bone
that does not have marrow (Hall, 2005c) so that a possible
marrow contamination is further eliminated. In addition, as the
only dermal skeletal elements in the trunk, and the first bone to
This article includes Supplementary Material available from the
authors upon request or via the Internet at http://
www.interscience.wiley.com/jpages/0021-9541/suppmat.
Contract grant sponsor: NHMRC, Australia.
*Correspondence to: Xin-Fu Zhou, DepartmentQ1 of CNS Trauma
& Rehabilitation, Research Institute of Surgery, Daping Hospital,
Chongqing 400042, P.R. China. E-mail: xin-fu.zhou@flinders.edu.au
Received 11 January 2007; Accepted 17 July 2007
Published online in Wiley InterScience
(www.interscience.wiley.com.), 00 Month 2007.
DOI: 10.1002/jcp.21251

ß 2 0 0 7 W I L E Y - L I S S , I N C .
2 LI AND ZHOU
ossify in rodents, the medial part cartilage of clavicle bone
and Kantomaa, 1988; Ronning et al., 1991; Rintala et al., 1996;
Author Proof
consists of actively dividing cartilaginous cells, histologically
resembling with the condylar cartilage of the mandible (Ronning
Hall, 2005c) are likely derivatives of neural crest (Graham et al.,
A
2004; Matsuoka et al., 2005; Hall, 2005c). Another distinct
feature of chondrocytes is their phenotypic instability in vitro as
chondrocytes have a propensity to de-differentiate into a
fibroblast-like phenotype (Schnabel et al., 2002; Vats et al.,
2005) and their de-differentiation or trans-differentiation may
occur easier than other adult committed cells.
Materials and Methods
Animals and reagents
Male and female Sprague–Dawley (SD) rats, 8–12 weeks old, were
used. All procedures were approved by Animal Welfare
Committee of Flinders University. Unless otherwise specified, all
reagents were of analytical grade and purchased from Sigma (St.
Louis, MO).
Explants culture of cartilage from adult clavicles
Animals were euthanased with an overdose of Halothane before
perfusion through the heart with cold sterile saline to flush the
blood cells from the system before tissue dissection at ambient
temperature (20–258C). The sternoclavicular joints were exposed,
and the medial part of the clavicles containing semi-transparent
cartilages was dissected out and put into culture dish. Cartilage
tissues were cleaned of any soft tissues under a dissecting
microscope. Isolated pieces of the cartilage tissue were pooled in a
Petri dish containing Hanks balanced salt solutions
without Caþþ, Mgþþ (D-HBSS, InvitrogenQ2) on ice, rinsed
extensively, and transferred to a separate Petri dish with air-
buffered Leibovitz-15 medium (Invitrogen). The tissue was then cut
into small pieces of about 1–2 mm3 blocks. After three washes with
L-15 medium, explants were suspended in the proliferation cell
medium (PCM) as described previously (Li et al., 2007). Clusters
and spheres combined with the free-floating single cells in the
explants flasks were collected every 3 days after initial culture, and
triturated with a fire-polished Pasteur pipette and the resulted cell
suspensions were passed through a 70-mm strainer (Falcon), and
then were reseeded at 100,000 cells per ml into additional tissue
culture flasks in PCM to exclude the explants. A half of the medium
was changed every 3 days and the changed medium was
centrifuged, filtered, and stored as the conditioned medium for
self-renewal analysis (see below). To improve cell expansion, some
culture flasks were tightly capped during incubation unless changing
medium, this procedure was expected to decrease the oxygen
level, which is shown to be beneficial for sphere forming and
maintenance of undifferentiated state (Morrison et al., 2000; Studer
et al., 2000; Lin et al., 2006).
Self-renewal assay
Spheres/clusters (primary) originally cultured in flasks without
explants were triturated with a fire-polished pipette, and the cells
were passed through a 40-mm cell strainer to create a single-cell
suspension. Cells were reseeded in cloning medium which consists
of PCM diluted 1:1 with the conditioned medium at a density of
5,000 cells per ml. After 7–10 day culture, some large size spheres
(secondary) were selected and dissociated by AccutmaxTM as
demonstrated by Wachs et al. (2003), the same low-density
methods (5,000 cells/ml) was employed for identification of the
formation of tertiary sphere to demonstrate the self-renewal
capability.
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
Differentiation and settings
For cloning identification. Single primary spheres were
placed on laminin-, ploy-L-lysine-, and fibronectin-coated coverslips
in 24-well plates or 8-well chamber Lab-Tech slides (Nunc), and was
covered with 20ml of DMEM/F12 (1:1) containing 15% fetal calf serum
(FCS, Invitrogen), and incubated for 30 min at incubator to let the
spheres/clusters attach on coverslips, then washed briefly once by
differentiation medium (DM, prepared similarly to PCM, omitted bFGF
and EGF but with 2 mM forskolin) with or without 2% FCS (as serum-
containing medium (SCM) or serum-free medium (SFM), respectively),
and then incubated in 500 ml of SCM or SFM for 2 weeks.
For growth factors-induced differentiation. Attached
secondary spheres on the coverslips were cultivated with DM in the
presence of 50 ng/ml recombinant human nerve growth factor (b-
NGF, Peprotech), or 50 ng/ml recombinant human glial growth factor 2
(GGF-2, Gift of Dr. Mark Marchionni, Cambridge NeuroScience, Inc.,
Cambridge, MA) for 1 week, and analyzed for their ability to
differentiate into neurons, glial cells, and other cells.
Immunocytochemistry
Sample preparation for immunostaining was performed as
previously (Li et al., 2007). Immunocytochemistry of cell cultures
and tissue sections was performed as described (Xian et al., 2004;
Chung et al., 2006; Lagares et al., 2007; Li et al., 2007) with slight
modifications. Samples were pre-incubated in blocking buffer
containing 5% donkey serum followed by overnight-incubation
with the primary antibodies and 2 h incubation with species-specific
secondary antibodies. Double-labeling or Triple-labeling
experiments were performed by simultaneously incubating
samples in appropriate combinations of primary antibodies
followed by non-cross-reactive secondary antibodies (Alexa-
conjugated secondary antibodies (1:500, Invitrogen) or Cy-
conjugated secondary antibodies (1:250–500, Jackson
ImmunoResearch LaboratoriesQ3)). The primary antibodies are
summarized in Table 1. In some samples, nuclei were
counterstained with 40 , 6-diamidino-2-phenylindole (DAPI). The
specificity of the light microscopic immunocytochemical
procedures was tested by omitting the primary antibodies, using
non-immune serum instead of the primary antibodies. The
procedures for BrdU labeling was based on protocols documented
by Valero et al. (2005) and by Tang et al. (2007). Anterior
subventricular zone, or intestines, dissected from BrdU injected
rats used as positive control tissues, and negative staining controls
consisted of staining sections from naı̈ve animals and staining of
experimental tissue with omission of primary antibody.
Autofluorescence, especially for explants samples, was quenched
by Sudan Black B or cupric sulfate as described previously (Brehmer
et al., 2004; Casella et al., 2004).
RNA extraction and semiquantitative reverse
transcription-polymerase chain reaction (RT-PCR)
Total RNA was isolated from the cultured secondary spheres,
acute isolated adult cartilage of clavicle, and positive controls
samples using TRI Reagent (Sigma) according to the supplier’s
protocol. Semiquantitative RT-PCR was performed on each gene
with triplication as described previously (Chie et al., 2001; Lagares
et al., 2007; Li et al., 2007). The protocol for the thermal cycler was
as follows: denaturation at 948C for 5 min, followed by 30–38
cycles of 948C (30 sec), optimal annealing temperature (see
Table 2, 1 min) and 728C (45 sec), with the reaction terminated by a
final 10-min incubation at 728C. Glyceraldehyde-3-phosphate
dehydrogenase (G3PDH) served as the internal control. Control
experiments without reverse transcriptase or without template
cDNA revealed no non-specific amplification. When PCR results
were negative, cDNAs from embryonic rat or BM-MSCs (for
details see Fig. 7 legend) were run as positive controls in parallel
with the negative samples to eliminate the possibility of false
negative results.
 N E U R O G E N I C P O T E N T I A L Q8 3

TABLE 1. Antibodies used, source, and dilution

Antigen (clone)

Nestin (RAT401)
BrdU (G3G4)
Type

IgG1
IgG1
Species

Mouse
Mouse
Author Proof
Clonality

Monoclonal
Monoclonal
Dilution

1:1,000
1:5,000
Source

Developmental Studies Hybridoma Bank

A
Collagen II (CIIC1) IgG2a Mouse Monoclonal 1:125
NF200 (N52) IgG1 Mouse Monoclonal 1:1,000 Sigma
b-III tubulin (SDL.3D10) IgG2b Mouse Monoclonal 1:500
Vimentin IgG Goat Polyclonal 1:1,000
SMA (1A4) IgG2a Mouse Monoclonal 1:500
GFAP IgG Rabbit Polyclonal 1:1,000 DAKO
p75NTR (MC192) IgG1 Mouse Monoclonal 1:1,000 Chemicon
Ki67 IgG Rabbit Polyclonal 1:1,000 Novocastra
Osteolcalcin IgG Rabbit Polyclonal 1:500 Dr. Fiona Zhou, Adelaide Uni., Adelaide, Australia
Runx2 IgG Rabbit Polyclonal 1:1,000
Collagen II IgG1 Rat Monoclonal 1:1,000 Dr. Cory Xian Adelaide Uni., Adelaide, Australia
Collagen X IgG Rabbit Polyclonal 1:500
p75NTR (9650) IgG Rabbit Polyclonal 1:2,000 Prof. M.V. Chao, NYU, NY
P0 (P05) IgG1 Mouse Monoclonal 1:500 Dr. J.J. Archelos, Graz Uni., Graz, Austria
TrkB IgG Chicken Polyclonal 1:3,000 Prof. L. Reichardt, UCSF, San Francisco

NF200, neurofilament 200; GFAP, glial fibrillary acidic protein; SMA, smooth muscle, a-actin; BrdU, 5-bromo-20 -deoxyuridine; p75NTR, p75 neurotrophin receptor; P0, Protein Zero.

In vivo BrdU labeling and in vitro chasing
To examine whether cells migrating out from explants are
proliferating cells in vivo and to exclude any possibility of other
possible non-chondrocytes migrating from in situ and then surviving
in our culture medium, we conducted in vivo BrdU labeling and
in vitro chasing test. Rats were intraperitoneally injected with BrdU
(50 mg/kg) every 12 h for 3 consecutive days. Twelve hours after
last injection, the clavicle cartilages were dissected and subjected to
explants cultured as above. The medial part of cartilages from
clavicles dissected from 4% paraformaldehyde perfused rats was
sampled for in situ BrdU detection. Cells migrating out from the
explants culture were used as cell-smear. The explants at different
time points were collected and samples prepared as above.
Cultured and uncultured cartilages were sectioned and stained for
BrdU and appropriate markers.
Image acquisition, cell counting, and statistical analysis
Stained samples were viewed under epifluorescent microscopy
using 10 0.3 NA, 20 0.5 NA, or 40 0.75 NA objectives
equipped with Phototmetrics CoolSNAP fx cooled CCD camera
on BX50 Olympus microscope with RS Image acquisition software.
The digitized images taken from epifluorescence microscope were
processed with NIH image for color recovery. Images were
adjusted for brightness and contrast, and assembled by Adobe
Photoshop 9.0 program; no other alterations were made. Cell
counts were performed by counting the number of positive-stained
cells using ImageJ program. Data are reported as a percentage of
total cells expressing positive marker labeling among total labeling
cells (XXXQ4200 cells) across three to four coverslips in triplicate
experiments. Statistical analysis for ANOVA or t-test was done by
SPSS 12.0 program. P < 0.05 was recognized for statistic
significance.
Results
Cells migrated from adult cartilages had
an ability to form spheres
In the culture with PCM, cells migrated out of the chopped
cartilage, and formed aggregates and spheres. We defined cell
aggregates that were loosely packed with irregular shapes as
clusters and sphere structures that were densely packed with a
round shape as spheres. As shown in Figure 1, 1 day after
culture (Fig. 1 upper part A), many cells migrated out and
floated in the medium as single cells or in clusters. One week
after culture (Fig. 1 upper part B), more clusters were seen and
cells were continuously budding out of cartilage tissues. Two
weeks after culture (Fig. 1 upper part C), many spheres could be
observed and these spheres resembled typical spherical
configuration (Fig. 1 upper part D). We defined spheres with
diameter larger than 200 mm as large spheres (Fig. 1 upper
part E). Cells were continuously budding around pieces of
cartilage tissues and more floating single cells and clusters were
observed in the culture. In addition to the floating cells, clusters
and spheres, some migratory cell could also attach on the
bottom of culture flasks and these cells were oval and rich in
matrix (arrowhead in Fig. 1 upper part F). Under the same
culture condition with time, the attached cells bore processes
(arrow in Fig. 1 upper part F), resembling the morphology of
neural cells.
Self-renewing capacity of sphere-forming cells
To see whether they were stem cell-like cells, we examined if
the sphere-forming cells had a self-renewal capacity. We hand-
picked large secondary spheres and dissociated the spheres and
cultured in low density. We counted the number of tertiary
spheres in the culture 2 weeks after plating. The result showed
that about 3.6% cells formed tertiary spheres and 0.23% were
large spheres (Fig. 1 lower part) under low-density culture.
About 30% of cells formed clusters containing a number of cells.
These results indicated that these cells had a capacity of self-
renewal and could be expanded in vitro.
Characterization of emigrating cells and spheres
Almost 100% cells emigrating from cartilage tissues after 3 days
in vitro explants culture were immunoreactive for RunX (Fig. 2,
lower parts A,B,C), a marker for chondroblasts (Goldring et al.,
2006), and collagen X (Green labeling in parts C,D of Fig. 3, and
part A in Fig. 4), a specific marker for hypertrophic
chondrocytes (Lefebvre and Smits, 2005; Goldring et al., 2006;
Westendorf, 2006). However, the immunoreactivities of
collagen II, the marker for mature hyaline chondrocytes
(Goldring et al., 2006), were relatively weak (Red in part D of
Fig. 3) and this is not due to methodological problems, as the
antibodies used here worked well in tissue sections
(Supplement Figs. 1 and 2). Thus, the emigrating cells are nearly
pure population based on Runx2 and collgen X labeling.
Majority of the cells were also osteocalcin-positive (Fig. 2,
upper part), a marker for late chondrocytic lineage cells and
oesteoblasts (Goldring et al., 2006) which are not recognized as
stem cell (Hall, 2005d). These data are consistent with the
notion that ‘‘hypertrophic chondrocytes can become
osteoprogenitor cells’’ (Hall, 2005b). Most of these cells also

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

TABLE 2. Primers and PCR conditions for respective genes

Gene Accession number and primers (5(–3() Annealing temperature (-C) Product size (bp) Region amplified

Oct 4

Sox 2
NM_001009178
5(-CTGCGGCCCCTGCTGGAGAAGT-3(
5(-TGGGGGCAGAGGAAAGGACACAGC-3(
XM_574919
5’-AGAACCCCAAGATGCACAAC-3’
5’-ATGTAGGTCTGCGAGCTGGT-3’
Author Proof 62

55
351

466
577–927

266–731

A
Sox 9 XM_343981 55 543 105–647
5(-GCTCGGAACTGTCTGGAAAC-3(
5(-TCCTCGCTCTCCTTCTTCAG-3(
Sox 10 NM_019193 56 439 520–948
5(-CAACCTCGGCGGCGGAAGAATG-3(
5(-GCCCGTAGCCAGCTGCCGAGTAG-3(
Notch1 XM_342392 55 364 8474–8837
5(-CTCACGCTGATGTCAATGCT-3(
5(-GTGGGAGACAGAGTGGGTGT-3(
Hes1 NM_024360 55 315 810–1134
5(-CTACCCCAGCCAGTGTCAAC-3(
5(-AAGCGGGTCACCTCGTTCAT-3(
Wnt1 XM_235639 61 383 1652–2034
5(-GTGCCCCCTCTCCCCGTGACCTCTC-3(
5(-GGCTGAAACCCCCGGCACAATAAAT-3(
Bmi-1 XM_229212 54 283 318–600
5(-ACTGGCCCCGTTTATCTCCTG-3(
5(-TGTTTGCCCACGGTTTTCTTTAT-3(
CD133 NM_021751 60 224 496–719
5(-TCATCCTGGGCCTGCTGTTCATTTT-3(
5(-GATCCGGGTCCTTGTCTGCTGGTT-3(
CXCR4 NM_022205 55 397 338–734
5(-GCCATGGCTGACTGGTACTT-3(
5(-TGGAGTGTGACAGCTTGGAG-3(
EdnrB NM_017333 60 565 1055–1619
5(-TTACAAGACAGCCAAAGACT-3(
5(-CACGATGAGGACAATGAGAT-3(
Krox-20 NM_053633 56 292 922–1213
5(-CACCACTTCCACCTCCTCTC-3(
5(-CTCACCGCCTCCACTTGCCC-3(
Olig1 NM_021770 55 471 1135–1605
5(-CCACCATAAGCTCACCCAGT-3(
5(-GCGAGCCTGAAAGACAGAAC-3(
Pax6 NM_013001 58 202 755–956
5(-AGTTCTTCGCAACCTGGCTA-3(
5(-GGAGCTGATGGAGTTGGTGT-3(
Msi-1 AY043393 55 304 249–552
5(-GAGCTCGACTCCAAAACAAT-3(
5(-AGCTTTCTTGCATTCCACCA-3(
NeuroD AF107728 62 413 249–661
5(-ACGGGGCCCCAAAAAGAAAAAGATGACC-3(
5(-AGGAGTAGGGATGCACCGGGAAGGAAGC-3(
Mash1 NM_022384 52 340 1036–1375
5(-GGCTCAACTTCAGTGGCTTC-3(
5(-TCGGAGGAGTAGGACGAAAC-3(
REST NM_031788 57 496 1061–1556
5(-ACAACGGGCCTAAACCTCTT-3(
5(-TCTGCGTTGTTTCCTGTCTG-3(
coREST NM_001013994 57 407 1251–1657
5(-GAGCATGAAGCAGACCAACA-3(
5(-GGGGTACTGATCGGGGTACT-3(
Id2 NM_013060 56 207 129–335
5(-CTCCAAGCTCAAGGAACTGG-3(
5(-ATGCTGATGTCCGTGTTCAG-3(
Id4 NM_175582 54 209 262–470
5(-GTCAGCAAAGTGGAGATCCTG-3(
5(-CTGTCGCCCTGCTTGTTCAC-3(
BDNF NM_012513 55 335 260–594
5(-AGGACGCGGACTTGTACACT-3(
5(-GCAGCCTTCCTTCGTGTAAC-3(
LC3 NM_199500 56 217 255–471
5(-GCCTGTCCTGGATAAGACC-3(
5(-TTGGGAGGCATAGACCATGT-3(
Beclin 1 AY033824 56 431 878–1308
5(-GTGCTCCTGTGGAATGGAAT-3(
5(-CCACTTGAGATTCGTCAGCA-3(
Col2a1 NM_012929 54 306 3100–3405
5(-AAGGGTGATCGTGGTGAGAC-3(
5(-AGGGCCAGAAGTACCCTGAT-3(
Col10a1 XM_001053056 54 333 846–1178
5(-TGCTAACCAGGGGGTAACAG-3(
5(-AGCCACACCTGGTCATTTTC-3(
G3PDH X02231 55 452 591–1042
5(-ACCACAGTCCATGCCATCAC-3(
5(-TCCACCACCCTGTTGCTGTA-3(

Oct4, octamer-binding transcription factor 4; Sox, SRY (sex determining region of Y-chromosome) -related HMG-box gene; Hes, hairy and enhancer of split 1; Wnt1, wingless-type MMTV
integration site family, member 1; Bmi-1, B lymphoma Mo-MLV insertion region 1; CXCR4, chemokine, CXC motif, receptor 4; EdnrB, endothelin receptor, type B; Krox-20, for example, EGR2
(early growth response 2); Pax6, paired box gene 6; olig1, oligodendrocyte lineage transcription factor 1; Msi-1, musashi-1, Drosophila, homolog of; NeuroD, neurogenic differentiation 1; Mash1,
mammalian achaete-scute homolog 1; REST, RE (repressor element) -1 silencer transcription factor; coREST, REST corepressor; Id, inhibitor of differentiation; BDNF, brain-derived
neurotrophic factor; LC3, microtubule-associated protein 1 light chain 3; Col2a1, procollagen, type II, alpha 1; Col10a1, procollagen, type X, alpha 1; G3PDH, glyceraldehyde-3-phosphate
dehydrogenase.
 N E U R O G E N I C P O T E N T I A L Q8 5

Author Proof

A
Fig. 1. Phase contrast photographs of cells emigrating out from cartilage and formation of spheres. Upper part: The phase contrast
microphotographs show that the cells migrated out from cartilage explants changed with time at 1 day (A), 1 week (B), 2 weeks (C) after in vitro
culture. The sizes of these multicellular aggregates vary from several cells to large solid spheres (arrows in C). The large size spheres could be
formed as shown in D and E after 2 weeks (D) and 4 weeks (F) in culture. The cells attached on the bottom of uncoated Flasks during explants culture
were stained by Commassie-Blue; F shows the different morphological profiles with process-bearing cells (arrows) or round/oval cells (arrowhead).
Lower part: cloning analysis of cartilage-derived spheres. Tertiary spheres were formed from dissociated cells of secondary spheres. The low-
density (5,000 cells/ml) culture was employed to identify the clone-forming ability of secondary spheres. The number of tertiary spheres was
counted in triplicate experiments and the sphere-forming efficiency (% of total cells) was calculated against the total number of cells seeded initially
and the result is shown in the upper table. The number of large size spheres whose diameter was more than 200 mm was also counted. A: a typical
phase-contrast micrograph shows tertiary spheres (arrowheads). Cell aggregates as indicated by large arrows were excluded from counting. Small
and thin arrows indicate floating single cells. B: a phase-contrast image shows a typical large-sized tertiary sphere with ‘‘budding-like’’ cells around
its perimeter as indicated by arrows.

expressed vimentin (Fig. 2, lower parts D,E,F), which

contributes to the maintenance of the chondrocytic phenotype
(Blain et al., 2006) and is also a useful marker for neural
progenitors derived from MSCs (Chu et al., 2006). To see
whether these sphere-forming cells expressed markers of
NSC, we labeled the emigrating cells with nestin (Lendahl et al.,
1990) and p75NTR (Stemple and Anderson, 1992). The result
showed that a subpopulation of cells form 1-day culture
expressed nestin (Fig. 2 upper part), more nestin positive
phenotypes appeared in 3 days culture (Fig. 3 part B, Fig. 4 part
A), and the ratio of nestin expressing cells further increased in
the subsequent culture (Fig. 4 part B) and passage (Supplement
COLOR

JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

6 LI AND ZHOU

Author Proof

A
Fig. 2. Primary cluster/spheresderived from adult claviclecartilageexpress markers for osteobalst, chondroblast and neural crest stem cells. The
upper part: a same micrograph field shows that representative primary clusters from 1-day explants culture expressed osteoblast marker,
osteolcalcin (A), or nestin (B, arrow). DAPI counter-staining was shown in (C). D: a merged view of parts (A,B,C). The arrow indicates nestin
positive cells. The lower part: the cluster and single cells from 3-days explants culture expressed Runx2 (A), the primary spheres also were positive
for vimentin (D) or p75NTR (G). Nuclei were counterstained with DAPI (B,E,H). C,F,I: respective merged views of parts (A,D,G).

Fig. 3). Interestingly, with time following the culture, another

useful marker for neural crest, p75NTR (Stemple and
Anderson, 1992; Morrison et al., 1999) was also expressed in
3-day culture and fully co-existed with nestin (parts B,C in
Fig. 3), the majority of sphere-forming cells in primary spheres
expressed p75NTR (Fig. 2, lower parts E,F), indicating that a
subpupolation of emigrating cells probably acquired phenotype
of neural crest stem cells in explants culture.
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JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP

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Fig. 3. Characterization of primary clusters/single cells using specific markers for chondrocytes (collagen II and Collagen X) and neural crest stem
cells (Nestin and p75NTR) by double-immunolabeling: part (A) is the control images showing staining-specificity which using normal IgG to replace
primary antibodies. Parts (B,C,D) show the representative microphotographs of emigrating cells/clusters double-labeled by nestin R p75NTR (B),
p75NTR R ColX, and ColII R ColX, respectively. Nuclei were counterstained with DAPI. ColII, Collagen II; ColX, Collagen X.

We next asked if the phenotypic changes were dynamic after

emigrating from explants, we compared the expression of
collagen X and nestin in free-floating cells at different time
points after emigration. As shown in Figure 3, the collagen X
expression of emigrating cells declined with time, on the
contrary, the ratio of nestin positive cells at 10 days in vitro was
remarkably higher than that at 3 days after emigration (part C in
Fig. 3), the same results were also observed in the passaged
culture (Supplement Fig. 4); Moreover, following these possible
phenotypic alterations, the emigrating cells did not show any
impairment of proliferation as demonstrated by Ki67þ cells
(Supplement Fig. 4) and neurosphere formation (Supplement
Fig. 5). The above data indicates that emigrating cells from adult
clavicle change their phenotypes in vitro, especially, acquire
some features of NSCs in the present settings.
We further conducted the double-staining of secondary and
tertiary spheres by the commonly used neural crest stem cell
markers, p75NTR and nestin. The majority of cells in the
secondary spheres were positive for nestin and p75NTR. Both
these markers were colocalized (Supplement Fig. 5A–D).
Comparing with the primary spheres/clusters, the number of
nestin positive cells was significantly increased in the secondary
spheres (data not shown). A similar result was observed in the
tertiary spheres. As shown in Supplement Figure 5E,F, almost all
cells in the tertiary spheres were positive for nestin and
p75NTR and they were highly colocalized. The data suggest that
the cells migrating out from cartilage tissues change their
phenotypes under the culture conditions. They were probably
converted back to neural crest stem cell-like cells with the time
and passages.

Characterization of the cells under

differentiation conditions
To characterize the cells under differentiating conditions,
primary and secondary spheres and clusters grew on laminin
and poly-D-lysine-coated surface upon the withdrawal of
mitogens FGF and EGF in serum containing medium. Cells were
stained for markers of neurons, glia, and smooth muscle cells,
the derivatives of neural crest. As shown in Figure 5, cells
differentiated into both neurofilament positive or beta III
tubulin positive neurons and GFAP positive glia (Fig. 5A,C).
Some differentiated cells could be stained with an SMA antibody
(Fig. 5B,D), a marker for myofibroblast (Stemple and Anderson,
1992; Morrison et al., 1999). Thus, the spheres derived from
adult cartilages could differentiate into cells with ectodermal or
mesodermal origin. Vimentin was expressed in most GFAPþ
and SMAþ cells from primary spheres after culture for 2 weeks
(Fig. 5A,C). More cells expressed immature neuronal marker
bIII-tubulin, GFAP, and SMA in secondary spheres after culture
for 2 weeks (Fig. 5B,D). We compared percentage of neural
(NF200þ or bIII-tubulinþ and GFAPþ) differentiation cells
between primary and secondary spheres with statistical
analysis. Group data indicated that the percentage of neural cell
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Fig. 4. Upregulation of nestin and downregulation of Collagen X in the emigrating cluster/cells with time following cultures: parts (A,B), the
representative microphotographs double-labeled by nestin (red) and collagen X (green), were taken from samples after 3-day (A) and 10-day (B)
culture excluded from explants. All the nuclei were counterstained with DAPI. C: bar graphs represent means W SEM, MMP < 0.01.

(NF200þ and GFAPþ) increased in the secondary spheres

comparing with those in the primary spheres (Fig. 4E). Some
cells in short-term (1 day) differentiating samples using
emigrating cells were co-labeled with the chondrocyte-specific
marker, collagen II and glial cell marker GFAP, suggesting that
these cells were probably intermediate transitional cells
(Supplement Fig. 6).
Differentiation under the challenge of growth factors
To test, whether these spheres respond to environmental cues,
we cultured these cells in DM in the presence of NGF or GGF-2,
respectively. As shown in Figure 6, among the neural lineage,
about 30% were neuron and 70% glia under the DM conditions
(Fig. 6A,D). NGF significantly increased the percentage of glial
lineage and decreased the percentage of neuron lineage. In
contrast, GGF increased percentage of neuron lineage but
decreased glial lineage. In addition, some differentiating cells
also display the possible functional neural phenotypes
(Supplement Fig. 7), which are positive for P0, marker for
myelin-forming cells (Archelos et al., 1993; Peirano et al., 2000),
and TrkB, the functional receptor for brain-derived
neurotrophin factor mediated myelination (Cheng et al., 2006).
Gene expression profile
We further selected a set of genes to explore molecule
mechanisms underlying this conversion by RT-PCR, as shown in
Figure 7 (lane 1 is mRNA from adult cartilage and lane 2 is
mRNA extracted from secondary spheres). No embryonic
stem cells marker Oct4 was detected in both tissues. There
were no detectable levels of CD133, a marker for
hematopoietic stem cells (HSCs), MSCs, and some NSCs. Both
of the chondrocytic marker genes, collagen II alpha 1 (colIIa1)
and collagen X alpha 1 (colXa1), and the chondrocytes-specific
transcriptional factors, sox9, were markedly downregulated in
the spheres, but proneural transcription factors, members of
the basic helix-loop-helix family members, olig1, mash1,
NeuroD, and RNA binding protein Musashi-1, and paired-box
transcription factor Pax6, inhibitors of differentiation (Id2 and
Id4) were upregulated in the secondary spheres. The genes
involving migration CXCR4, and endothelin receptor B, and the
genes related to myelination sox10 and krox20 were up
regulated. The expression of the genes for autophagy, Beclin1,
and MAP-LC3, were also remarkably altered.

In vivo labeling and in vitro chasing

To further validate that the migratory cells were proliferating
chondrocytes, we performed in vivo BrdU labeling of
chondrocytes and in vitro chasing experiments. We label
proliferating chondrocytes by BrdU pulsing in the adult rats
before explants culture in vitro. As shown in Figure 8,
chondrocytes in adult cartilage were positive for BrdU and
Ki67, a cell proliferation-associated antigen (Schluter et al.,
1993), and after culture in vitro, they migrated out, and nearly
100% free-floating cells were BrdUþ and Ki67þ positive
chondrocytes. We also found that the majority of cells
accumulated in the margin of explants clavicle cartilage are
collagen X labeling cells with BrdU incorporation (Supplement
Fig. 2), this is consistent with the observation that the majority
of free-floating cells after 3 days explants culture is
hypertrophic chondrocytes (parts C,D in Fig. 3 and part A in
Fig. 4).

Discussion
The aim of the present study was to determine whether a
committed and differentiated adult chondrocytes can be
converted into neurogenic cells with potential of self-renewal
and multipotential differentiation under a standard NSC culture
condition in vitro. We demonstrated that clavicle cartilage-
derived chondrocytes robustly migrated out of the explants,
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Fig. 5. Multipotency and neural differentiation of the primary (A,B) and secondary (C,D) spheres in serum-containing medium. The
differentiation profiles were determined by double- or triple-immunostaining for lineage specific markers, NF200 or bIII-tubulin, GFAP, SMA, and
vimentin. E shows percentages of neural cell lineages (NF200R or GFAPR) from primary and secondary spheres after culture for 2 weeks. Bar
graphs represent means W SEM (MMP < 0.01).

and formed spherical structures by the sphere culture

technique (Reynolds and Rietze, 2005), and illustrated that the
sphere-forming cells, at least, had a limited self-renewing
capacity and differentiated into neuronal, glial and smooth
muscle cell lineages. In accordance with the de-/trans-
differentiation properties, a set of genes specific to cartilage
chondrocytes was downregulated and the other set of genes
related to neural crest progenitors were upregulated. NGF and
GGF were able to alter the differentiation behaviors by
increasing glial and neuronal differentiation, respectively. Thus,
this study provides evidence that fully committed chondrocytes
can de-/trans-differentiate to neural crest stem-like cells.

Isolation and purification of chondrocytes

from adult cartilage tissues
Compared with widely used dissociation culture, the explants
culture has an advantage of mobilizing proliferating
chondrocytes by virtue of their intrinsic migratory and chemo-
attractive features. Based on the known feature of cartilage
tissues which do not contain cells from nerves, blood,
connective tissues and blood vessels, the cells migrating out
from the tissue are likely homogenous chondrocytes. The
immunochemical analysis showed that almost 100% cells
migrating out from pieces of clavicle cartilage were
immunoreactive for ColX and Runx2, the cartilage
chondrocyte and chondroblast markers (Lefebvre and Smits,
2005; Goldring et al., 2006; Westendorf, 2006), supporting the
homogenous nature of cartilage chondrocytes. Further
evidence comes from our in vivo labeling and in vitro chasing
experiments. It is known that cartilage chondrocytes are
proliferating cells and highly instable cell lineage (Tallheden
et al., 2003; Goldring et al., 2006). We took the advantage of
in vivo proliferation of chondrocytes and analyzed the nature of
emigrating cells by injecting rats with BrdU 3 days earlier. Our
data showed that the BrdU positive cells in the cartilage had
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Fig. 6. Effects of NGF and GGF2 on the differentiation of cartilage-derived sphere-forming cells. After culture for 1 week in specific medium, cells
were stained for NF200 (red) and GFAP (green), and counterstained with DAPI. A: DM only; (B) with NGF; (C) with GGF2. Arrows in (A,C) show the
cells (yellow color) double-stained by NF200 and GFAP simultaneously. D: group data show the effect of NGF and GGF on the percentages of
NF200R and GFAPR cells. Data are expressed as means W SEM from triplicate experiments in parallel cultures and analyzed by ANOVA and
student’s t-test (MMP < 0.01, compared with DM group only; ##P < 0.01 compared with NGF group).

typical morphology of chondrocytes. Most floating cells from

the BrdU labeled cartilage tissues in vitro were BrdU positive and
Ki67 positive. This experiment further supports that the
emigrating cells from cartilage explants were predominantly
proliferating hypertrophic chondrocytes. Thus, we conclude
that sphere-initiating cells mainly originated from chondrocytes
which acquired phenotype of neural crest-like progenitor cells
under the culture conditions. Further evidence supporting this
note was the finding that there is a decline of collagen X positive
cells with an increase of nestin expressing cells in the
subsequent cultures, and there are more p75NTRþ/nestinþ
cells in the tertiary spheres. More importantly, the progressive
loss of chondrocytic markers (Collagen II and Collagen X) and
transcriptional factor Sox 9 was accompanied with the
upregulation of neurogenic genes without impairment of
proliferation capability, suggesting a conversion process.
However, our study could not absolutely exclude the possible
existence of a very rare number of intrinsic neural crest stem
cells in the cartilage which could concomitantly emigrated
under the culture condition. The supposed reserve cells should
retain some primitive markers, such as Oct4 (Nichols, 1998;
Loh et al., 2006), and should not possess committed cells labels,
such as collagen X and Runx2, however, this is not the case in
the present study. Although the neural crest markers, p75NTR
and nestin, are immunohistochemically detected in a
subpopulation of cells in the clavicle cartilage in situ (data not
shown), these p75NTRþ/nestinþ cells clearly displayed hyaline
morphology. It is possible that p75NTRþ/nestinþ
chondrocytes are more likely to be converted into neural crest
progenitors under the certain conditions. This issue will be the
topic of future study.

Mechanisms underlying the conversion from

chondrocytes to neural crest stem cell-like cells
The major finding of our present study was that chondrocytes
derived from clavicle cartilage were able to reverse their
phenotype, self-renew and differentiate into ectodermal and
mesodermal lineage cells through the generation of neural crest
cells with stem cell properties in vitro. De-differentiation and
trans-differentiation are most likely mechanisms underlying the
conversion. Under the standard NSC culture medium used by
other researchers (Morrison et al., 1999; Kruger et al., 2002),
chondrocytes become neural crest progenitors at first, then
ectodermal lineage cells and mesodermal lineage cells. Several
lines of evidence from our present study support this notion.
First, we chose the unique cartilage tissue as our model to
reduce the possibility of contamination of other stem cells such
as MSCs and adipocyte stem cells, which are known to have a
potential of neural differentiation (Baksh et al., 2004). Thus, it is
unlikely that the NSC-like cells identified in the present study
are contaminated MSC cells or HSC cells. Lack of CD133 gene,
the marker for MSC and HSC, in our preparation also supports
this notion. Secondly, migratory cells from cartilage can form
self-renewal spheres with potential of differentiation into
neuronal, glial, and smooth muscle cell lineages. This
characteristic was mostly likely acquired in vitro via the induction
because the adult clavicle cartilage chondrocytes do not
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Fig. 7. Comparison of gene expression profiles between adult cartilage tissues and adult cartilage-derived secondary spheres by RT-PCR analysis.
Left part: The representative band of the PCR products running in 1.7% agarose gel electrophoresis. Rve: positive controls: cDNA from E14 rat
embryo for Oct4 and Sox2, cDNA from adult rat bone marrow stem cell for CD133, CXCR4, and Bmi-1, respectively. Lane 1 adult cartilage, lane 2:
adult cartilage derived secondary spheres. G3PDH was used as an internal control. Right part: Tabularized results of RT-PCR for the tested genes in
total RNA isolated from adult cartilage of clavicle and adult cartilage-derived secondary spheres. () No signal; (W) minor signal; (R) weak signal;
(RR) moderate signal; (RRR) strong signal. Abbreviations: see Table 2.

express neuronal markers and smooth muscle markers in vivo

(data not shown). Thirdly, the comparison of the gene
expression profiles between clavicle cartilage and the cartilage-
derived spheres supports the conversion hypothesis. Lack of
primitive stem cell gene such as Oct-4 in parental cartilage
suggest the sphere forming cells were not reserved primitive
stem cells (Young, 2004) in the niche. The upregulation of stem
cell genes (Masashi1, NeuroD, Olig1, krox20 and sox10) and
concomitant downregulation of cartilage specific genes (colIIa1,
cloXa1, and sox9) in spheres suggest sphere-forming cells
(chondrocytic origin) changed their phenotypes into neural
crest progenitors. Downregulation of neural repressor/
silencer genes REST and co-REST in these spheres comparing
with parental tissues also indicates their neurogenic potential in
the culture. Finally, we have detected a small potion of
emigrating cells simultaneously expressed both chondrocytic
marker collagen II (Lee et al., 2004; Goldring et al., 2006), and
glial cell marker GFAP (Supplement Fig. 7). The presence of
these intermediate transitional cells under the in vitro induction
condition indicates a dynamic phenotype switch, a sign for
trans-differentiation occurred in the process of stem cell
plasticity (Steindler, 2006).
Alternatively, the explants cultures could be assumed as a
pathological setting. Cells under this process need to change
their metabolism rapidly in response to a changing
environment, and increase the turnover of long-lived proteins
via the autophagy mechanism, the non-ubiquitin-proteasome
protein degradation system for cell homeostasis. This protein
degradation mechanism mainly functions as the effective
recycling and turnover of cytoplasmic constituents of
eukaryotic cells (Mizushima et al., 2004; Hara et al., 2006;
Komatsu et al., 2006), and has been integrated into various
developmental and physiological events, including embryonic
development (Qu et al., 2007), remodeling of cells (Juhasz and

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Fig. 8. In vivo labeling of chondrocytes with BrdU and in vitro identification of emigrating cells. A–I: Are microphotographs of clavicle cartilage
explants sections from rats injected withBrdUand stained for BrdU (red)and Ki67 (green). A–C: 1 dayin vitro; (D–F) 3 days invitro; (G–I) floating cells
collected at 3 days from explants culture. C,F,J: Are merged images of (A,B), (D,E), and (G,H), respectively. Inset in (F) is the enlarged
microphotograph showing the region marked with the box in (F). Arrows in (A–C) indicate double labeled (BrdUR/Ki67R) cells; the arrowheads in
(A–C) indicate cells of BrdUR/Ki67; arrows and arrowheads in (G–I) mark cells of Ki67R/BrdUR and BrdUKi67R, respectively. These double-
and/or single-labeled cells indicate they may exist in explants or in suspensions at different cell-cycle phases. J: is a histogram showing percentage of
BrdU positive and negative cells in the total of ki67 labeling cells as shown in (G–I), data are expressed as means W SEM from triplicate experiments
in parallel cultures.

Neufeld, 2006), even cell fate decision (Lavieu et al., 2007).

Beclin1 and MAP-LC3, markers of this process (Yue et al., 2003;
Kouno et al., 2005), were upregulated in the present study,
indicating self-clearing process is active and reprogramming
may occur in our explants culture.
Chondrocytes are developmentally derived from either
mesenchymal cells or neural crest (Hall, 2005a,c). It has been
argued that the presence of resident stem cells in adult tissues
may represent re-programming of cells under in vitro culture
and other pathological conditions (Anderson, 2001; Young,
2004). It is possible that the postmitotic cell reprogram, which
occur in amphibian (Brockes and Kumar, 2002), rejuvenate
(Conboy et al., 2005) to acquire phenotypic features of their
late ancestor, for example, neural crest cells. An earlier report
showed that articular chondrocytes from human can be
reprogrammed into primitive cells and these cells have same
characteristics to MSCs by analysis of morphology and cell
surface markers in vitro (de la Fuente et al., 2004). However, our
present data argue against the conversion to MSC but support
for the neural crest cell model. Our preparation did not express
MSC specific genes such as prominin-1 (CD133) and Oct-4.
Furthermore, the morphology and sphere-forming behavior of
clavicle derived NSC-like cells are different from that of MSC,
which are adherent and fibroblast-like in shape. Nevertheless,
our present study support the unifying hypothesis that adult
stem cells derived peripheral tissues are all progeny of the
neural crest (Pierret et al., 2006), which are generated via the
mechanisms of de-differentiation and trans-differentiation or
the reprogramming process. Further examples supporting this
hypothesis are stem cells recently isolated from fetal rat calvaria
(Zhang et al., 2006), adult cornea (Yoshida et al., 2006), adult rat
dorsal root ganglia (Li et al., 2007) and adult rat trigeminal
ganglia (Lagares et al., 2007), all of which are the derivatives of
neural crest.
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P75NTR is a marker associated with the embryonic (Stemple
and Anderson, 1992; Rao and Anderson, 1997) and adult
(Kruger et al., 2002) neural crest stem cells, and the p75NTR-
positive MSC from adult bone marrow are clonogernic
precursors, which have a greater capacity to give rise to
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multipotent differentiation (Quirici et al., 2002). We found that
a small subpopulation of free-floating collagen X positive cells
from clavicle explants after 3 days culture expresses p75NTR,
and its expression significantly increases following subsequent
culture, thus it is possible that p75NTR plays an import role in
the acquirement of multipotential differentiation and in
conferring these cells responsiveness to neurotrophins which
result in diverse phenotype acquisition.
Factors in the culture medium may conduct
the trans-differentiation
In the present study, different growth factors and chemicals
present in the culture medium may play critical roles for the
trans-differentiation of chondrocytes. In addition to the
neurobasal medium with N2 and B27 supplement, the critical
epigenetic molecules in our chemically-defined medium which
might contribute to this conversion are probably bFGF and
retinoic acid (RA), both of which are routinely used in the
expansion of mammalian neural crest stem cells isolated from
embryonic sciatic nerve (Stemple and Anderson, 1992;
Morrison et al., 1999) or adult gut (Kruger et al., 2002). Basic
FGF produces neurogenic, proliferative, and patterning effects
on adult CNS stem cells (Dono, 2003; Cayuso and Marti, 2005).
RA is a commonly used neural induction reagent in vitro
(McCaffery et al., 2003; Mey and McCaffery, 2004) and enhances
expression of markers of neural crest (Kawaguchi et al., 2005)
and stimulates de-differentiation of articular chondrocytes
(Hering et al., 2004). However, it is unlikely that b-
mercaptoethanol and DMSO (final concentration: 0.1%, used as
the dissolve reagent for b-mercaptoethanol 1,000 stock)
present in the culture medium played any role in the conversion
in our preparation as the concentrations used were too low to
cause the reported artifact (Lu et al., 2004; Neuhuber et al.,
2004; Bertani et al., 2005), and the omission of these chemicals
reproduced the same results.
Neural crest progenitors exhibit a cell-intrinsic bias in
responses to instructive factors that influence the outcome of
lineage decision. Our results showed that GGF-2 induced more
cells expressing neuronal markers than NGF, whereas NGF
promoted glial differentiation. These results are surprising as it
is well recognized that GGF promotes gliogenesis (Falls, 2003;
Jessen and Mirsky, 2005). In the neural crest stem cell cultures
from embryonic sciatic nerve, GGF-2 inhibits the development
of neurons (Shah et al., 1994). Our results, however, argue
against the anti-neurogenic role of GGF-2. The mechanisms
underlying the pro-neurogenic effect of GGF-2 in our
preparations are not known. Different neural crest
progenitors, such as chondrocyte origin, may generate the
opposing results. GGF-2 may exert its role by promoting
neuronal survival and proliferation. It is known that GGF-2
promotes the survival, migration, and proliferation of nascent
trunk neural crest cells (Bannerman et al., 2000), neural crest-
derived precursor cells (Jessen and Mirsky, 2005), or the
differentiating neurons (Liu et al., 2005). In contrast to GGF-2,
NGF increased the proportion of glial cells in the differentiation
assay. The mechanism underlying the action of NGF is not
known. Since p75NTR, the receptor for NGF, is present in
these cells, it is possible that NGF preferentially induced
apoptosis of neuronal and non-neuronal cells (lacking of its high-
affinity receptor TrkA) via the p75NTR (Miller and Kaplan,
2001), and thus NGF treatment probably resulted in relatively
increased proportion of glia cells with low-levels of p75NTR.
This is the case in the nervous system during development
JOURNAL OF CELLULAR PHYSIOLOGY DOI 10.1002/JCP
13
where NGF could cause programmed cell death of
subpopulation of neurons mediated by p75NTR (Bibel and
Barde, 2000).
The present study is significant in several aspects. Firstly, we
have provided a clear example of trans-differentiation
mechanisms from a committed and differentiated cell type to a
NSC-like progenitor, using clavicle chondrocytes as a model.
Our example contributes to the argument that committed cells
can be reversed to their progenitors with capacities of self-
renewing and trans-differentiation into lineages of different
developmental origins. Secondly, we first report that neural
crest stem cell-like cells can be efficiently generated from
cartilage cell via our unique method of explants culture where
actively proliferating chondrocytes and/or neural crest cells can
break the barrier of dense extracellular matrix (ECM) including
collagen fibers and proteoglycans, and emigrate out with
unknown mechanisms. The rigid ECM, which is also important
for attachment and cell migration, perhaps modified by
emigrating cells through changed expression of remodeling
molecules. The molecules such as mitogens and retinoic acid in
the medium could induce a set of transcriptional program which
underlies the motility capacity of chondrocytes, such as
upregulation of genes related to migration, for example,
CXCR4 (Kucia et al., 2004), endothelin B receptor (Barlow
et al., 2003; Kruger et al., 2003), sox10 (Hong and Saint-Jeannet,
2005), as well as p75NTR (Yamauchi et al., 2004). Additional
candidates involved in the cellular motility are enzymes related
to matrix remodeling, as well as signaling molecules related to
the process of migration of adult chondrocytes, which should
contain ECM-degrading enzymes, such as matrix
metalloproteinases (Yong, 2005; Malemud, 2006; VanSaun and
Matrisian, 2006; Page-McCaw et al., 2007; Pizzi and Crowe,
2007) and their regulators. Our method of using biological
property of neural crest cells to isolate stem cells has the
advantages of simplicity, highly purity, economy, and efficiency
over the dissociation method. Thirdly, the neural crest stem
cell-like cells from clavicle cartilage may be a suitable, accessible
source for therapeutic significance for the repair of injured
central nervous system. Clavicle cartilage has been used as graft
to replace damaged human temporomandibular joint (Hall,
2005c), thus, it is possible to remove a piece of clavicle cartilage
from patients without affecting the motor function and to
generate NSCs for autologous transplantation. Harnessing
trans-differentiated/de-differentiated cells as a therapeutic
modality will complement the use of committed cells in the
treatment of degenerative disorders (Burke and Tosh, 2005;
Kindler, 2005).
Acknowledgments
The authors are grateful to Prof. M.V. Chao (New York
University) for p75NTR antibody (9650), Dr. Fiona Zhou and
Dr. Cory Xian (Adelaide University, Adelaide, Australia) for
Collagen II, Collagen X, osteolcalcin, and Runx2 antibodies, and
Dr. Mark Marchionni (Cambridge NeuroScience, Inc.,
Cambridge, MA) for rhGGF2 protein; we thank Prof. Ian
Gibbins (Flinders University of South Australia) for help with
image acquisition, Mr. John Wang, Ms. J.X. Mi, Ms. Jinhua Zhong,
and Dr. Yongjun Fan in our laboratory for technical assistance.
The Monoclonal antibodies obtained from the Developmental
Studies Hybridoma Bank under the auspices of NICHID and
maintained by The University of Iowa, Department of Biological
Sciences, Iowa City, IA 52242.
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