Introduction

• Oligodendrocyte precursor cells (OPC) can behave as multipotent neural stem-like

cells (NSLCs) when the cells are cultured in a specific condition, whether the
committed myelin-expressing, late-stage oligodendrocytes, resembling OPC, can be
reprogrammed into NSLCs is still unknown.

• It has been demonstrated epigenetic modification of chromatin structure that results

in expression silencing/onset of specific genes underlies this reprogramming process, as
well as cell fate determination and identity during the directed differentiation of
neural stem cell. It will be also of great interests that if more neurons can be acquired
from oligodendrocytes under conditions of global epigenetic modulation through
histone de-acetylation inhibition by valproic acid (VPA), and/or through DNA
methylation inhibition by 5-aza-2-deoxycytidine (5azaD).
Materials & Methods
 Results
• OLN-93 cells change their phenotypes in PCM.
Fig.1: OLN-93 change
their sheet-like profile
of in SCM (A) into
polar-like morphology
(B) as revealed by
A B p75NTR staining. The
remarkably down-
regulation of myelin
basic protein during
PCM culture (C), and
accompanying
degrades MBP and
active proliferation
shown by BrdU
C D
incorporation (D).
 Characterization of
spheres/clusters
derived from OLN-93
A B C
• Fig.2: Characterization of
OLN-93 derived spheres
D /clusters by ICC. After 2
weeks in PCM, the
suspend spheres and
cluster appeared (A). The
E sphere-forming cells
express Nestin (B,E),
Ki67(B), p75NTR(D),
F
NG2(E),Nkx2.2 and TrkA
(G). Some cells are
positive for bipotent OPC
marker A2B5 (C), and the
G majority of cells
incorporates BrdU (D,F).
Self-renewal of sphere-forming cells

The low-density (5000 cells/ml) culture was employed to identify the clone-forming ability of the
primary, third, and seventh spheres, respectively. The number of the subsequent 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 mean ±
SEM. Left column shows the typical phase-contrast micrographs.
A
Characterization of differentiation

• Fig3. Muitipotent differentiation of OLN-

93-derived spheres following serum or
growth factors challenging. The upper
panel (A) is the representative
microphotographs showing the
B differentiation of primary spheres/cluster
under serum-induced differentiation. A
subpopulation of cells are positive for
neuronal markers NF200, beta III-tubulin,
as well as doublecortin (DCX). A small
portion shows co-labelling with glial
(GFAP+) and neuronal (NF200). With a
two-week challenge of a combination of
growth factors (50ng/ml, per factor), the
differentiated cells from some clones
showing typical neuronal and glial
phenotypes as revealed by a set of
markers including NF200 and DCX (for
neurons), S100 and GFAP (for
astrocytes), p25 and RIP (for
oligodendrocytes), as well as nestin. The
representative images were presented in
the lower panel (B).
 The effects of genetic remodelling inhibitors on the
cell fate determination of reprogrammed cells

B C D
E
• Fig.4: VPA, and/or 5azaD, single or combined administration, has different effects on the
acquisition of cell fate. Three-day insults by VPA (2mM) and/or 5azaD (5µM) following
subsequent 2-week differentiation, the samples were double-stained by NF200 and GFAP, the
representative microphotographs under different treatment were marked by A (DM-only, the left
three images are the same field labelling), B (+VPA), C (+5azaD), and D (VPA & 5azaD),
respectively. Scale bar: 50µM. E shows percentages of NF200+ or GFAP+ cells after culture
for two weeks. Bar graphs represent means ± SEM from triplicate experiments in parallel
cultures (**, ## p<0.01, compared with DM treatment, t test, ).
 Gene-expression profile after treatment
of chromatin remodelling factors

• Fig.5: Comparison of gene

expression profiles by RT-PCR
analysis. Upper panel (A): The
representative band of the PCR
products running in 1.7% agarose
gel electrophoresis. Lanes from
left to right as the marked. G3PDH
was used as an internal control.
Lower panel (B): Tabularized
results of RT-PCR for the tested
genes by arbitrary unit based on
the comparison with DM-only
samples. “↔”: no change; “↓↓↓,
↓↓, ↓” shows the amplitude of the
down-regulated genes from
“remarkable, moderate , minor”,
respectively. Otherwise, “ ↑↑↑, ↑↑,
↑” marks the up-regulated genes.
Abbreviations: see table 2.
 Analysis of signalling pathways alteration after
treatment of chromatin remodelling factors

• Fig. 6: Possible involvement of ERK

and Akt pathway but not CREB and
STAT3 in the cell-fate alteration
induced by VPA and/or 5azaD.
Protein samples isolated from sphere-
differentiation culture in the absence
(DM) or presence (+) of VPA (2mM)
and/or 5azaD (5µM) for three days
were immunoblotted by the marked
antibodies. Left column: Western
blots analysis of the same samples
with phospho-antibodies (top, each
panel) and non-phospho- antibody
(middle), and the stripped membranes
were then re-probed by tubulin (E7,
lower) as internal and loading control
.Right column shows densitometric
analysis of the data (normalized by
internal control) representing mean ±
s.e.m. from three or more sets of
samples immunoblotted in duplicates
on two gels as presented in the left
column (**, p<0.01 compared with DM
). Note that VPA suppresses total Erk
and pErk, and 5azaD reduces the
level of pAKt. However, both of them
have no significant effects on CREB
and STAT3 and their phosphorylation.
 Conclusions
• Myelin-expressing immature oligodendrocytes, resembling with OPCs, could
be reprogrammed into NSLCs characterized by expression of specific stem
cell markers, multipotent differentiation with self-renewal capacity.
•
Global histone modifications produce profound influences on the re-
differentiation of reprogrammed cells which VPA prefer to coax the neuronal
differentiation, and 5azaD result in astrocyte acquisition; The changes of
specific genes expression and phosphorylation of signaling molecules may
be underpinned these modulations.

•Acknowledgements
The authors are grateful to Prof. C Richter-Landsberg for OLN-93 cell line,
Prof MV Chao (New York University, New York) for p75NTR antibody, and Dr.
Mark Marchionni (Cambridge NeuroScience, Inc., Cambridge, MA) for rhGGF2
protein. Some Mabs were obtained from the DSHB under the auspices of
NICHID and maintained by The University of Iowa, IA 52242. This work was
supported by grants from NHMRC of Australia (160052, 229961).