Dedifferentiation of Lineage-Committed Cells by a Small Molecule

Article abstract of DOI:10.1021/ja037390k

Combinatorial libraries were screened for molecules that induce mouse myogenic lineage committed cells to dedifferentiate in vitro. A 2,6-disubstituted purine, reversine, was discovered that induces lineage reversal of C2C12 cells to become multipotent pr

Full text of DOI:10.1021/ja037390k

Published on Web 12/19/2003
Dedifferentiation of Lineage-Committed Cells by a Small Molecule
Shuibing Chen, Qisheng Zhang, Xu Wu, Peter G. Schultz,* and Sheng Ding*
Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute,
10550 North Torrey Pines Road, La Jolla, California 92037
Received July 18, 2003; E-mail: sding@scripps.edu; schultz@scripps.edu
Among organisms that have regenerative capabilities, the urodele
amphibians are unique in their use of a cellular dedifferentiation
mechanism1 at the damaged site to form a blastema which contains
dedifferentiated progenitor cells. These cells can proliferate and
redifferentiate under developmental control to regenerate a wide
variety of tissues, such as limbs, tails, and lens. In mammals,
epimorphic regeneration is largely limited by an irreversible
differentiation process. As a consequence, stem cells,2 in particular
embryonic stem cells (ESCs) which can be expanded indefinitely
and are pluripotent, have attracted considerable attention as a
therapeutic approach to the damage caused by cardiovascular
disease, neurodegenerative disease, diabetes and aging. However,
the use of stem cells in cell replacement therapy remains problem-
atic for a number of reasons, including the lack of robust methods
for the propagation and efficient differentiation of stem cells, as
well as host rejection of allogeneic cells. The ability to dediffer-
entiate or reverse lineage-committed cells to multipotent progenitor
cells might overcome many of these obstacles. With an efficient
dedifferentiation process, it is conceivable that healthy, abundant,
and easily accessible adult cells could be used to generate different
types of functional cells for repair of damaged tissues. Moreover,
recent studies of the plasticity of murine myotubes3,4 and other cells
derived from adult tissues suggest that dedifferentiation may be
possible in mammalian system.5 However, in contrast to the
differentiation process, methods for the control and study of
dedifferentiation are lacking.
Cell-based phenotypic assays and, more recently, pathway
screens of synthetic small molecules and natural products have
historically provided very useful chemical probes of complex
cellular processes.6 The identification of small molecules which
induce dedifferentiation of somatic cells would help to elucidate
the molecular mechanism of this phenomenon and may ultimately
allow us to regenerate tissues in vivo. Toward this end, we have
begun to search for small molecules which can induce cellular
dedifferentiation of lineage-restricted mammalian cells. Although
little is known about the signaling pathways which might initiate
such a process, protein kinases are likely to be involved. Therefore,
libraries of heterocyclic compounds7 designed around a large
number of kinase-directed scaffolds were screened, including
substituted purines, pyrimidines, quinazolines, pyrazines, pyrrol-
opyrimidine, pyrazolopyrimidine, phthalazines, pyridazines, and
quinoxalines. Herein, we report the discovery of a small molecule
from those libraries that induces a dedifferentiated phenotype in
murine C2C12 myoblasts.
Figure 1. Reversine.
Scheme 1. Dedifferentiation Screening Scheme
into myotubes. However, myoseverine was found to bind micro-
tubules, and its effects most likely stem from cytoskeleton remodel-
ing rather than dedifferentiation. To identify molecules that induce
true dedifferentiation, an alternative assay was devised which is
based on the notion that lineage-reversed myoblasts should regain
multipotency. Specifically, they should acquire the ability to
differentiate into multiple nonpermitted cell lineages when exposed
to conditions that typically induce differentiation of multipotent
mesenchymal progenitor cells into adipocyptes, osteoblasts, or
chondrocytes. Osteoblast formation was chosen for the primary
screen since there are established osteogenic-inducing conditions
and a high throughput assay for detecting the bone specific marker,
alkaline phosphatase (ALP).9
A two-stage screening protocol (Scheme 1) was used in which
C2C12 cells were initially treated with a library of 50 000 discrete
small molecules for 4 days to induce dedifferentiation and then
assayed for their ability to undergo osteogenesis upon addition of
known osteogenic-inducing agents. To carry out the screen, C2C12
cells were plated in 384-well plates in growth medium (DMEM
with 10% fetal bovine serum) and after overnight incubation (during
which time cells attach to the bottom of the plate) 5 µM of
compound was added. After 4 days, compound was removed, and
the medium was changed to osteogenic-inducing medium10 contain-
ing 50 µg/mL ascorbic acid-2-phosphate, 0.1 µM dexamethasone,
and 10 mM â-glycerophosphate. The culture was maintained for
an additional 7 days, and cells were lysed and then assayed for
ALP activity using the fluorogenic substrate 2′-[2′-benzothiazoyl]-
6′-hydroxybenzothiazole phosphate (BBTP). Because this screen
could also find molecules which directly induce transdifferentiation
of myogenic cells to osteogenic cells, compounds from the primary
screen were tested to determine (1) whether they can induce
osteogenesis in the absence of the osteogenesis-inducing cocktail
and (2) whether cells treated with compounds can differentiate into
adipocytes under conditions11 that induce adipogenesis.
The murine C2C12 cell is a myogenic lineage committed
myoblast. Upon withdrawal of serum, confluent C2C12 cells can
differentiate and fuse into characteristic multinucleated myotubes.
We have previously shown that a small molecule, myoseverine,
has the ability to cleave multinucleated myotubes to generate
myoblast-like cells,8 which can be proliferated and redifferentiated
Among a series of 2,6-disubstituted purine analogues identified
in the primary screen, a 2-(4-morpholinoanilino)-6-cyclohexylami-
9
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10.1021/ja037390k CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
of ODM reversine alone has no osteogenesis activity, further
confirming that reversine induces dedifferentiation of C2C12 cells
rather than trandifferentiation to osteogenic lineage.
A preliminary structure-activity relationship (SAR) analysis of
the primary screen data revealed that both of the N9 hydrogen and
the NH substitution at the C2 position of the purine ring are essential
(removal of either completely abolishes activity). However, primary
amines at the C6 position of the purine ring can be replaced with
various heteroatoms, such as oxygen and sulfur without loss in
activity, suggesting a H-bond donor at this position is not required.
Only a limited group of aromatic substituents can be tolerated at
the C2 position of the purine ring. We are continuing to explore
the SAR in an effort to further enhance the potency and specificity
of reversine, as well as to identify a linkage position on the molecule
for attachment to a solid support for affinity experiments to elucidate
its cellular target(s).
In summary, we have discovered a 2,6-disubstituted purine,
reversine, which can induce myogenic lineage-committed cells to
become multipotent mesenchymal progenitor cells which can
proliferate and redifferentiate into bone and fat cells. This phe-
nomenon recapitulates key aspects of epimorphic regeneration.
Figure 2. Morphological and histological characterization of cellular
dedifferentiation induced by reversine. (A) Phase image of day 4 culture
after reversine treatment; (B) phase image of day 4 culture after DMSO
treatment; (C) ALP staining (red) of day 7 osteogenic differentiation culture
after reversine treatment; (D) Oil red O staining (red) of day 7 adipogenic
differentiation culture after reversine treatment; (E) ALP staining (red) of
day 7 osteogenic differentiation culture after DMSO treatment; (F) oil red
O staining (red) of day 7 adipogenic differentiation culture after DMSO
treatment.
Acknowledgment. Funding was provided by the Skaggs
Institute for Chemical Biology and the Novartis Research Founda-
tion (P.G.S. and S.D.). This is manuscript number 15951-CH of
The Scripps Research Institute.
Supporting Information Available: Detailed experimental pro-
cedures and spectra data of the compounds disclosed in this paper
(PDF). This material is available free of charge via the Internet at http://
pubs.acs.org.
nopurine analogue (which we named reversine, Figure 1) was found
to induce the highest level (7-fold) of ALP activity relative to the
DMSO control treatment. On day 4 of compound treatment, striking
differences were observed between the reversine-treated and
untreated cells. In the control cells (treated only with DMSO),
multinucleated myotubes were formed throughout the culture
(Figure 2B). In contrast, in the presence of 5 µM reversine myotube
formation was completely inhibited, and cells continued to grow
to form a confluent culture of mononucleated cells (Figure 2A). In
addition, myogenic-specific markers such as MyoD and myosin
began to disappear. These results suggest that reversine is not simply
acting as a selective toxin.12
After 4 days treatment, the compound was removed, and cells
were then grown in osteogenic differentiation medium (ODM) or
adipogenic differentiation medium (ADM). At the end of day 7,
under ODM conditions, 35% of cells stained positive for ALP
(Figure 2C); similarly when exposed to ADM condition, 40% of
cells had the characteristic fat cell morphology, oil droplets inside
the cytoplasmic membrane, and stained positive with oil red O
(Figure 2D). Again, in the control culture, confluent C2C12 cells
continue to form myotubes and were unaffected by the ODM and
ADM conditions (Figure 2E,F). These results clearly demonstrate
that reversine-treated lineage-committed C2C12 myoblasts cells
regain multipotency. Consistent with this observation, in the absence
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(12) At the effective concentration (1-10 µM), no significant cell death was
observed during dedifferentiation stage. In addition, transdifferentiation
of C2C12 myoblasts to osteoblasts or adipocytes was not observed under
the conditions used to induce osteogenesis or adipogenesis. These
observations suggest that reversine acts as a dedifferentiation-inducing
agent rather than simply enriching certain type of progenitor cells by killing
myoblasts.
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