Dedifferentiation? What’s next?
differentiate ESCs to specific ASCs; however, this would require a
Sungwoo Kim,1 Gus R. Rosania,2 and Young-Tae Chang3
1
Department of Immunology, The Scripps Research Institute,
La Jolla, CA 92037; 2Department of Pharmaceutical Sciences,
University of Michigan College of Pharmacy, Ann Arbor, MI
48109; 3 Department of Chemistry, New York University,
NY, NY 10003
Stem cells have elicited a great deal of attention because of their
enormous potential for tissue and cell replacement therapy.
Although embryonic stem cells (ESC) can differentiate into many
tissue types (i.e., pluripotent), direct injection of ESC into mice
induces benign tumors (1). In contrast, partially committed adult
stem cells (ASC) can only differentiate into certain lineages of cells
(i.e., multipotent) without resultant tumor formation, thus having
greater potential as therapeutic materials. Although the prepara-
tion of ESCs is a dramatic technical advancement––especially
those recently derived from human sources (2)––the low num-
ber of extant ASCs within a very large mixed population of cells
poses an even tougher challenge for collection and cultivation. To
surmount these difficulties, one approach would be to selectively
thorough understanding of the mechanisms regulating differentia-
tion. Alternatively, dedifferentiation of somatic cells into ASC-like
cells, followed by selective differentiation into another cell or
tissue, offers another route. Becuase it is generally believed that
differentiation is a unidirectional, nonreversible event, achieving
dedifferentiation is a major challenge.
Modulating the differentiation of ESCs or ASCs by using
small-molecule cocktails or by genetic modification has been
demonstrated, but most efforts have been devoted to driving
ESCs or ASCs down differentiation pathways by “natural” means,
for example, through the use of growth factors. In this context,
reversine, recently reported by Chen et al. (3) is a very interesting
small molecule. The authors added reversine to myoblast C2C12
cells that otherwise differentiate into multinucleated myotubes.
The reversine-treated cells became multipotent cells and could
be redirected to differentiate into osteoblasts or adipocytes using
a cell-specific differentiation cocktail: osteogenic differention
medium (ODM; ascorbic acid-2-phosphate, dexamethasone, and
β-glycerophosphate), or adipogenic differentiation medium [ADM;
3-isobutyl-1-methylxanthine, dexamethasone, and insulin or per-
oxisome proliferator–activated receptor gamma (PPARγ) agonist
April 2004
Volume 4, Issue 2 83
 Viewpoint
(personal communication with Sheng Ding, Scripps Institute)]
(Figure 1). These findings open up the possibility of a small-
molecule treatment that changes one cell-type into another. The
authors explain that the first step of reversine treatment induced
“dedifferentiation” of the muscle committed C2C12 cells into mul-
tipotent progenitor cells. The dedifferentiation hypothesis can be
one possible answer; however, other explanations could account
for this phenomenon.
Although C2C12 cells are considered to be a myogenic lin-
eage–committed cell line, it is known that treatment of C2C12
cells with bone morphogenic proteins (e.g., BMP-2) induces their
transdifferentiation into osteoblasts (4), and transfection of C2C12
cells with a dominant-negative form of T cell factor 4 (TCF4, a
transcription factor) converts C2C12 cells into adipocytes (5). It
seems that C2C12 myoblasts have the potential to direct their dif-
ferentiation to a variety of different cell types; this phenomenon
has been explained as transdifferentiation, which might be a
unique property of C2C12 cell lines in vitro, and might not nec-
essarily be a property of the natural muscle cells as they exist in
the body. According to the authors of the reversine study, C2C12
cells in growth media were treated with reversine for four days.
Under the same conditions, the authors report that untreated
(control) C2C12 cells started to express markers of differenti-
ated muscle cells, which can be induced if cell growth becomes
arrested because of contact inhibition or growth factor depriva-
tion. Thus, the activity ascribed to reversine can also be explained
if reversine inhibits the ability of C2C12 cells to differentiate into
muscle––without inhibiting their ability to differentiate into other
cell types. In other words, with reversine treatment, C2C12 might
Figure 1. Suggested pathway involving reversine-mediated dedifferentiation and subsequent
redifferentiation. See text. Insert: reversine’s structure. ADM, adipogenic differentiation medium;
ODM, osteogenic differentiation medium.
84
not differentiate into muscle; therefore, their capacity to differenti-
ate into other cell types is more visibly manifested.
To address these different possibilities, it will be key to
undertake a molecular mechanistic study to identify reversine’s
direct target. Additionally, more detailed analysis of the differen-
tiation pathways, perhaps by using cell markers of intermediate
differentiation stages, should also lead to a better understanding of
whether reversine acts via dedifferentiation or some other mecha-
nism. To fish out the target protein(s), efficient affinity matrix
preparation would be the most straightforward strategy. However,
anchoring biologically active small molecules to affinity matrices
through the use of molecular linkers could inhibit a small mole-
cule’s original activity. Thus, structure–activity relationship (SAR)
studies are essential for the proper design of small molecule-lin-
kere-matrix interactions. The authors described preliminary SAR
results, but none of the information provided gives an idea as to
where the linker could be attached. For example, the NH group at
the C6 position of the purine ring of reversine could be replaced
with an O or an S without loss of activity, which confirms that the
hydrogen donor at that position is not required, but the substitu-
tion of O or S would not allow for extra linker attachment sites.
More numerous SAR studies will be required to achieve an active
reversine affinity matrix. In fact, knowing where to add covalent
linkages is a significant problem in Forward Chemical Genetics (a
research approach starting from phenotype screening to leading to
target identification) and natural product chemistry in the target
identification step. Depending on the binding orientation of small
molecule to the target protein, the affinity matrix preparation pro-
cedure is at best cumbersome and sometimes completely impos-
sible. One possible solution is the
“tagged library approach,” whereby
all the library compounds are
prelabeled with a linker tag, and
the compounds are screened in the
presence of the tag moiety. Thus,
selected active tagged library com-
pounds will be directly connected
to the affinity moiety in order to
identify the biological target pro-
teins, thus eliminating the need for
a further SAR study by utilizing
the already existing tag (6).
Although it will be neces-
sary to identify reversine’s direct
mechanism of action to determine
whether reversine is really the
dedifferentiation-inducing mol-
ecule that it is claimed to be, other
aspects of reversine that also need
to be urgently addressed are its
cell- and tissue-type specificity and
applicability to differentiated cells
obtained for actual tissues. C2C12 cells are not the equivalent Sungwoo Kim, PhD, is a Research
of differentiated muscle cells, in vivo or in vitro. Thus, whereas Associate in the Department
reversing differentiation of C2C12 cells growing may be fascinat- of Immunology at The Scripps
ing, reversing the differentiation of a cell obtained directly from Research Institute. His research
muscle tissue of a human patient is an even greater challenge. focuses on oncogenic signaling
Is reversine effective only in myoblasts? Or can reversine act on pathways.
other cell types? For example, can reversine change adipocytes or
bone cells to muscle cells, thus confirming that its activity is really
“dedifferentiation?” If reversine’s activity is indeed dedifferentia-
tion, if it works in primary, differentiated muscle cells, and if it
happens to be myoblast specific, the search for small molecule
Gus R. Rosania, PhD, is
dedifferentiators of other cell types will be keeping scientists in
an Assistant Professor of
this field busy for many years to come. DOI: 10.1124/mi.4.2.5
Pharmaceutical Sciences and
Member of the Center for
References
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a practical technique for the preparation of human ESCs.
3. Chen, S., Zhang, Q., Wu, X., Schultz, P.G., and Ding, S.
Dedifferentiation of lineage-commited cells by a small molecule. J. Am. Young-Tae Chang, PhD, is an
Chem. Soc. 126, 410–411 (2004). Assistant Professor of Department
4. Lee, M.H., Kim, Y.J., Kim, H.J. et al. BMP-2-induced Runx2 expression is of Chemistry at New York
mediated by Dlx5, and TGF-β1 opposes the BMP-2–induced osteoblast
differentiation by suppression of Dlx5 expression. J. Biol. Chem. 278,
University. Dr. Chang’s research
34387–34394 (2003). projects are designed to combine
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Erickson, R.L., MacDougald, O.A. Inhibition of adipogenesis by Wnt sig- with high-throughput screen-
naling. Science 289, 950–953 (2000).
ing and molecular evolution. His
6. Khersonsky, S.M., Jung, D.W., Kang, T.W. et al. Facilitated forward
chemical genetics using tagged triazine library and zebrafish embryo laboratory is also interested in
screening. J. Am. Chem. Soc. 125, 11804–11805 (2003). This article the development of new biologi-
was the first to describe a tagged library approach for facilitated
cally active molecules through the development and screening of
target identification.
combinatorial libraries. Address correspondence to YTC. Email
yt.chang@nyu.edu; fax 212-260-7905.

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