CHEMICAL BIOLOGY
Small Molecules to Control Stem Cell Fates
Simon Hilcove, Yue Xu and Sheng Ding, The Scripps Research Institute, Department of Chemistry and the Skaggs Institute for Chemical Biology, La Jolla, California
doi: 10.1002/9780470048672.wecb559
Stem cells hold great promise for the treatment of many devastating diseases and will also provide new insights into the molecular mechanisms that control developmental and regenerative processes. Realization of the therapeutic potential of stem cells will require a better understanding of the signaling pathways that control stem cell fate as well as an improved ability to manipulate stem cell proliferation, differentiation and reprogramming. Cell-based phenotypic and pathway screens of synthetic compounds have led recently to the discovery of several small molecules that can be used to control stem cell fate. Such molecules will not only provide new insights into stem cell biology but will also facilitate the development of therapeutic agents for regenerative medicine.
Given appropriate conditions, stem cells can self-renew for long periods of time while maintaining the ability to differentiate into various functional cell types in the body (1). It is these characteristics that not only make stem cells a useful system in which to study tissue and organ development but also give them great potential for regenerative medicine. Given the success of well-practiced, cell-based therapies (e.g., hematopoietic stem cell transplantation for treating hematological diseases and pancreatic islet cell transplantation for type I diabetes), it is conceivable that this approach could be applied to many other serious medical conditions where cells are lost because of disease, injury, or aging. Current challenges in cell replacement therapy include the limited source of engraftable stem/progenitor cells and a poor ability to manipulate their functional expansion and differentiation. Alternatively, drug stimulation of the body’s own regenerative capabilities (i.e., proliferation, differentiation, migration, or even reprogramming of endogenous cells to replace the damaged cells/structures) may represent a more desirable therapeutic approach to fulfilling the ultimate goal of regenerative medicine. Although tremendous efforts have been put into these areas, it is clear that a better understanding of stem cell biology is required before these approaches can be realized.
Small molecules have long been associated with biological discoveries. Our understanding of biological processes often develops from discovering or designing ways to perturb a given process and observing the effects of the perturbation. Although genetic approaches have been widely used for this purpose, the small-molecule approach clearly offers some distinct advantages. For example, small molecules provide a high degree of temporal control over protein function, which generally acts within minutes or even seconds, and their effects are often reversible, which facilitates both rapid inhibition and activation. Their effect can also be finely tuned by varying concentrations of the compound of interest. Moreover, because of the inherent difficulty of genetic manipulation for many types of stem cells (e.g., low transfection efficiency or poor clonal expansion), small-molecule tools are especially useful for the stem cell field. In this article, recent developments in the use of small molecules (Fig. 1) on the various aspects of stem cell biology such as self-renewal, differentiation, and reprogramming will be reviewed.
Figure 1. Chemicals that control stem cell fate.
Biological Background of Stem Cells
Stem cells normally are classified, based on their origin and differentiation capacity, as either embryonic or adult stem cells (1). Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst. ESCs can self-renew indefinitely and are pluripotent—(the ability to differentiate into all cell types in the embryo proper). Adult stem cells are undifferentiated (unspecialized) cells that are found in differentiated, or specialized, tissue. They have limited self-renewal capability and generally can only differentiate into the specialized cell types of the tissue in which they reside. These cells function as the “reservoir” for cell/tissue renewal during normal homeostasis or tissue regeneration. Sources of adult stem cells have been found in most tissues, including bone marrow, blood stream, cornea and retina of the eye, dental pulp of the tooth, liver, skin, gastrointestinal tract, lung, pancreas, heart, and brain. For example, the bone marrow is a mesoderm-derived tissue that consists of a complex hematopoietic cellular system supported by stromal cells embedded in a complex extracellular matrix. A growing body of evidence suggests that bone marrow contains at least two types of stem cells: hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), both of which are multipotent. HSCs have the capacity to provide life-long reconstitution of all blood-cell lineages after transplantation, whereas MSCs have the ability to differentiate, both in vivo and in vitro, into a variety of adult mesenchymal cell types, including osteoblasts, chondrocytes, and adipocytes.
Stem cell fate in vivo is under strict control from both intrinsic and extrinsic factors, and loss of this control has been postulated to be a key step in degenerative and carcinogenic processes (2). Emerging evidence suggests cancer initiation results from an accumulation of oncogenic mutations (intrinsic loss of control) in long-lived stem cells or their immediate progenitors, followed by modification of the surrounding microenvironment (loss of extrinsic control) (3). Cancer stem cells have been detected in leukemia, breast, and brain tumors (4-7). They may originate from resident stem cells or develop as a result of a gain of self-renewal capacity in tissue progenitor cells. Therefore, the characterization of a cancer stem cell profile within diverse cancer types, and a better understanding of the biology of its counterpart, the normal stem cell, may open up new avenues for cancer treatment.
Stem cell expansion and differentiation ex vivo commonly are controlled by culturing cells in a specific configuration (e.g., an attached monolayer or as suspended aggregates) with cocktails of growth factors and signaling molecules as well as genetic manipulations. However, most of these conditions are either incompletely defined or are nonspecific and inefficient at regulating the desired cellular process. Such conditions often result in inconsistency in cell culture and mixed populations of cells that would not be useful in studying specific cellular processes or in cell-based therapies. More efficient and selective conditions for homogeneous stem cell self-renewal and differentiation into specific cell types need to be developed before the various applications of stem cells can be realized. Towards this end, chemical approaches serve as excellent tools to investigate the underlying mechanism and to control the specific stem cell fate.
Chemical Approaches to Stem Cell Research
Conceptually, two approaches exist for discovering chemical compounds in stem cell biology. The target-based approach involves development of chemical compounds for specific biological targets and the application of these compounds in order to link their targets’ modulation to the produced pharmacologic effect in cells or organisms. In the phenotype-based approach, small-molecule libraries are screened in high-throughput functional assays (in cells or whole organisms) to identify compounds that produce a desired phenotype, followed by elucidation of the molecular targets or pathways that they engage.
The success of both target-based and phenotype-based methods relies heavily on the qualities of the chemical libraries used. Although combinatorial technologies allow the synthesis of a large number of molecules with immense structural diversity, it is impossible to saturate the chemical space, which has been estimated to contain more than 1060 molecules (8). Because biological space interacts with only a fraction of chemical space, synthetic attempts to increase randomly the molecular diversity of a chemical library by introducing a high level of structural variability/complexity drastically reduce the libraries’ fitness to a given biological selection/screen, which results in most molecules being inactive. Furthermore, screening of even larger chemical collections to increase the chance of finding hits can compromise assay functionality, sensitivity, and fidelity for practical reasons (9). As the diversity and fitness of a library can be conflicting goals, the careful design of a chemical library becomes a critical aspect of combinatorial synthesis.
The notion of “privileged structures” describes selected structural motifs that can provide potent and selective ligands for biological targets (10). Privileged structures typically exhibit a greater tendency of interacting with biological targets and good “drug-like” properties (11, 12). One of the most straightforward and productive ways to generate “privileged” chemical libraries is to use key biological recognition motifs as the diversity elements for combinatorial synthesis (13). In this approach, a variety of naturally occurring and synthetic heterocycles that are known to interact with proteins involved in cell signaling (e.g., kinases, cell surface and nuclear receptors, or enzymes) are used as the core molecular scaffolds. Then, a variety of substituents can be introduced into each of these scaffolds to create a diverse chemical library. Using this method, a diverse heterocycle library that consists of over 100,000 discrete small molecules (representing over 30 distinct structural classes) was generated with an average purity >90%, which has proven to be a rich source of biologically active small molecules targeting various proteins involved in a variety of signaling pathways.
The screening method is another important factor for the phenotypic approach. Although the use of simple reporter systems or enzymatic activity assays would allow higher throughput in cell-based assays, the monitoring of more complex phenotypic changes (e.g., cell morphology, multiple biomarker expression and localization, and cell physiology) by high content imaging methods substantially reduces false analysis from primary screens and provides broader assay versatility (14, 15). Furthermore, informatics on the matrix of assays/compounds/genes has facilitated bioactive compound identification and deconvolution of their mechanism.
Small-Molecule Regulators of Stem Cell Fate
Self-renewal is the process by which a stem cell divides to generate one (asymmetric division) or two (symmetric division) daughter stem cells with identical developmental potentials as the mother cell (16). The self-renewal ability of stem cells is central to development and the maintenance of adult tissues.
Self-renewal of stem cells can be regarded as a combined phenotypic outcome of cellular proliferation and inhibition of differentiation and cell death. Consistent with this notion are the findings that self-renewal of murine embryonic stem (mES) cells can be achieved in the absence of feeder cells and serum in a chemically defined media condition by the combined activity of two key signaling molecules: LIF/interleukin 6 (IL6) family members and bone morphogenic protein (BMP) (17). LIF activates STAT (signal transduction and activation of transcription) signaling through a membrane-bound gp130-LIFR (LIF receptor) complex to promote proliferation and to inhibit mesoderm and endoderm differentiations of mES cells via a Myc-dependent mechanism. BMP induces expression of Id (inhibitor of differentiation) genes via Smad signaling and inhibits differentiation of mES cells to neuroectoderm.
To gain a better understanding of ESC self-renewal, a cell-based screen for small molecules that can promote mESC self-renewal was carried out using an established Oct4-GFP reporter mESC line and examining pluripotency marker expression (Oct4) as well as morphological analysis (undifferentiated ESCs having compact colony morphology). A novel, synthetic small molecule named pluripotin was discovered in this screen that is sufficient to propagate mESCs in the pluripotent state under chemically defined conditions in the absence of feeder cells, serum, and LIF. Long-term cultures of mESCs with pluripotin can be differentiated into cells in the three primary germ layers in vitro, and they can also generate chimeric mice and contribute to the germ line in vivo. Affinity chromatography using a pluripotin-immobilized matrix identified ERK1 and RasGAP as the molecular targets of pluripotin. Additional biochemical and genetic experiments confirm that pluripotin is a dual-function, small-molecule inhibitor of both ERK1 and RasGAP, and that simultaneous inhibition of both protein activities is necessary and sufficient for pluripotin’s effects on mESCs. Because pluripotin’s mechanism of action is independent of LIF, BMP, and Wnt signaling, this study suggests that ES cells may possess the intrinsic ability to self-renew and that inhibition of differentiation pathways would be sufficient for maintaining the undifferentiated state. Thus, not only did the discovery of pluriotin provide interesting insights to the mechanism of ESC self-renewal, but it also exemplified the power of small-molecule screens in that it is possible to modulate more than one target by a single small molecule to achieve a desired complex biological phenotype. In addition to the unbiased phenotypic screen described above, based on known mechanistic insights, p38 (e.g., SB203580), MEK (e.g., PD98059), or GSK3 (e.g., BIO) inhibitors have been shown to enhance self-renewal of mESCs in the presence of additional signaling inputs (18-20).
Consistent with the self-renewal model, recent studies on Notch signaling exemplified the contribution of cell survival signaling to stem cell self-renewal. Androutsellis-Theotokis et al. (19) found that activation of Notch promotes fetal neural stem cell (NSC) survival, most likely through activation of AKT, STAT3, and mTor; this survival signal is antagonized by JAK and p38 MAPK, and applying JAK and p38 inhibitors significantly improves survival of NSCs. Interestingly, this mechanism also functions in human ESCs. Moreover, p38 inhibitors have also been shown by another independent study to promote HSC life span, which confirms even more the role of p38 in stem cell self-renewal (21). Although the details of the molecular mechanism still need to be worked out, these inhibitors clearly have had a beneficial effect on stem cell culture.
Most stem cells can divide via either asymmetric or symmetric modes (22). Because each asymmetric division generates one daughter with a stem-cell fate (self-renewal) and one daughter that differentiates, it was postulated that asymmetric division could be a barrier to stem cell expansion. Therefore, conversion of asymmetric division to symmetric division would promote self-renewal and long-term culture of stem cells. Indeed, this idea was confirmed by using one small molecule, the purine nucleoside xanthosine (Xs) (23). This small molecule promotes guanine ribonucleotide biosynthesis that reversibly converts cells from asymmetric division kinetics to symmetric division kinetics. It was found that Xs derived from stem cell lines exhibit Xs-dependent symmetric kinetics, and this derived stable line shows enhanced self-renewal. This study underscores the importance of balance between the two modes of division, both to stem cell expansion and to the regenerative capacity of adult stem cells.
One major challenge in hESC culture is a low survival rate after cell dissociation. Genetic manipulation (e.g., gene-targeting), and to a lesser extent, routine culture and directed differentiation are all reliant on clonal survival and/or cell dissociation. In a small screen, a selective small-molecule inhibitor of p160-rho-associated coiled-coil kinase (ROCK), Y-27632, was found to increase hESC survival. The mechanism of action by which Y-27632 inhibits apoptosis, which is yet to be identified, should yield insights as to the causes of poor survival after dissociation (24).
Recent studies have identified multipotent isl1+ cardiovascular progenitors (MICPs) in mouse embryos, and they have also been cloned from mESCs (25). MICPs can produce the three main cell types of the heart: cardiac muscle, smooth muscle, and endothelial cells. To gain insights into the pathways and mechanisms that regulate the self-renewal and differentiation of MICPs, a high-throughput screen was completed to identify small molecules capable of inducing expansion of isl-1+ MICPs (26). Several molecules were identified that significantly increased MICP expansion, with the potent GSK-3beta inhibitor BIO being one of the strongest. Furthermore, replacement of BIO and CMCs with a Wnt3a-producing feeder layer also facilitated MICP expansion, whereas treatment with Dickkopf-1 (DKK-1), which is an extracellular inhibitor of Wnt signaling, decreased the number of isl-1+ progenitor cells. Interestingly, BIO treatment also induced expansion of human isl1+ progenitors, which suggests a conserved role of Wnt signaling in self-renewal of MICPs.
Lineage-specific differentiation
Differentiation is the developmental process by which early pluripotent cells acquire the features of late-stage, mature cells such as neurons, hepatocytes, or heart muscle cells. Currently, few examples of devised, highly selective, and efficient conditions for stem cell differentiation into specific homogeneous cell types have been reported because of a lack of understanding of stem cell signaling at the molecular level. Small-molecule phenotypic screens provide another means to generate desired cell types in a controlled manner. Several small molecules have been identified by this method that modulate specific differentiation pathways of embryonic or adult stem cells.
Neural and neuronal differentiation
Retinoic acid (RA), forskolin, and HDAC inhibitors have been shown/used to induce neuronal differentiation of hippocampal adult neural progenitor cells. However, these factors are either pleiotropic or of undefined physiologic relevance: RA has neuronal subtype patterning activity and also been reported to induce cardiac differentiation (27) as well as pancreatic differentiation (28); HDAC inhibitors are nonspecific; and forskolin activates protein kinase A and serves to increase the cellular levels of the general signaling molecule cAMP (29).
Neuropathiazol, which is a substituted 4-aminothiazole, was identified recently from a high-content imaging-based screen of chemical libraries that specifically induces neuronal differentiation of multipotent adult hippocampal neural progenitor cells (30). More than 90% of the neural progenitor cells treated with neuropathiazol differentiated into neuronal cells as determined by immunostaining with βIII tubulin and the characteristic neuronal morphology. Interestingly, neuropathiazol can also inhibit astroglial differentiation induced by LIF and BMP2, whereas RA cannot, which suggests that neuropathiazol functions by a different mechanism and has more specific neurogenic inducing activity. The precise molecular target(s) of neuropathiazol is still unknown, although its identification will surely provide mechanistic insights into neuronal differentiation. Additionally, neuropathiazol can be used as a specific inducer to generate homogeneous neuronal cells, and it may serve as a starting point for development of small-molecule drugs to stimulate in vivo neurogenesis.
In an elegantly devised approach, functional motor neurons were generated from mESCs by sequential treatments with RA (neuralizing and caudalizing mESCs) and a specific small-molecule agonist (Hh-Ag1.3) of Hedgehog (Hh) signaling (ventralizing the caudalized cells) (31). This experiment underscores the importance of following developmental progression through sequential induction and combinatorial factors for generating a late-stage cell type from early stem cells.
Differentiation of mesenchymal stem/progenitor cells
Mesenchymal stem cells (MSCs) are capable of differentiating into all mesenchymal cell lineages, such as bone, cartilage, adipose, and muscle, and play important roles in tissue repair and regeneration. Many small molecules have been used to control the differentiation of mesenchymal stem/progenitor cells to a variety of cell types. For example, 5-aza-C (a DNA demethylation agent) can induce C3H10T1/2 cells (a mouse mesenchymal progenitor cell line) to differentiate into myoblasts, osteoblasts, adipocytes, and chondrocytes by enhancing cell differentiation competence via epigenetic modifications. Dexamethasone (a glucocorticoid receptor agonist) is another kind of epigenetic modifier, and its combination with other small molecules, such as ascorbic acid, β-glycerophosphate, isobutylmethylxanthine (IBMX, a nonspecific phosphodiesterase inhibitor), or peroxisome proliferator-activated receptor γ (PPARγ) agonists (such as rosiglitazone) have been used widely to modulate osteogenesis or adipogenesis of MSCs under specific conditions (32, 33). To identify small molecules that selectively induce osteogenesis of MSCs, a high-throughput screen of chemical libraries in C3H10T1/2 cells using an enzymatic assay of alkaline phosphotase (a specific osteoblast marker) led to the identification of a novel synthetic small molecule, purmorphamine (13). Genome-wide expression profiling in conjunction with systematic pathway analysis were used to reveal that the Hh signaling pathway is the primary affected biological network by purmorphamine (34). Additional studies, including chemical epistasis using known Hh pathway antagonists (cyclopamine and forskolin), have confirmed that purmorphamine’s mechanism of action on osteogenesis is through specific activation of the Hh pathway, and it acts at the level of Smoothened (Smo) (35). More recently, it was shown that purmorphamine directly targets the protein Smoothened (Smo) through competitive displacement assays using fluorescently-tagged cyclopamine.
Recently, a small-molecule screen completed in zebra fish embryos identified prostaglandin E2 (PGE2) synthesis as a novel modulator of vertebrate HSC function (30). Molecules that up regulated PGE2 synthesis increased HSC function, whereas those that lowered PG synthesis decreased HSC function. Moreover, treatment with a stabilized 16,16-dimethyl derivative of PGE2 improved kidney marrow recovery after irradiation in the adult zebra fish. Furthermore, PGE2 also induced an amplification of multipotent progenitors derived from mESCs, which demonstrates a conserved role in vertebrate species. These studies suggest that modulation of the PGE2 pathway could potentially be used to treat patients undergoing bone marrow transplant or to treat anemia.
Regeneration
Terminally differentiated, post-mitotic mammalian cells are thought to have little or no regenerative capacity, as they are already committed to their final specialized form and function and have permanently exited the cell cycle (36). However, it is conceivable that appropriate stimulation of mature cells to re-enter the cell cycle and proliferate may provide new therapeutic approaches for treating various degenerative diseases and injuries. The mammalian cardiomyocyte is one such mature cell type that has attracted substantial research efforts toward its regeneration. Using a target-based approach, a p38 MAPK inhibitor, SB203580, and a GSK3 inhibitor, BIO, have been shown independently to promote proliferation in both neonatal and adult cardiomyocytes indicated by BrdU incorporation and histone 3 phosphorylation (37, 38). The proliferation in adult cardiomyocytes was also observed to be associated with transient dedifferentiation of the contractile apparatus. Activation of canonical Wnt signaling by BIO also promotes proliferation of cardiac progenitor cells. The pancreatic β cell is another highly sought cell type for regeneration, the transplantation of which, in conjunction with simultaneous prevention of their immune destruction, may represent a “cure” for type 1 diabetes. Recent phenotypic screens of large chemical libraries have identified several classes of small molecules that can promote proliferation of human β cells, among which p38 inhibition was identified as the mechanism of action for one class of molecules. A major challenge lying ahead for proliferation of mature cell types is that the process typically is associated with loss of the cell phenotype (e.g., proliferated β cells undergo eptithelial-to-mesenchymal transition (EMT) to acquire fibroblast-like features, and they typically do not redifferentiate back to the mature β cells). Strategies for inducing functional redifferentiation of the proliferated cells or for inhibiting loss of cell identity while proliferating are highly desirable and are under intense investigations. It should be noted that a synthetic purine analog, myoseverin, was identified previously from a phenotypic screen, which can induce cleavage of multinucleated myotubes to generate myoblast-like cells, which can proliferate and redifferentiate into myotubes (39).
In the mammalian CNS, failure of axonal regeneration is attributed not only to the intrinsic regenerative incompetence of mature neurons but also to the inhibitory actions of CNS myelin and molecules in the glial scar at the lesion sites. In a cell-based screen to identify small molecules that can counteract the inhibitory activity of CNS myelin, several EGFR inhibitors (including PD168393 and AG1478) were found to promote neurite outgrowth of cerebellar granule neurons on an immobilized myelin substrate (40). More importantly, PD168393 and Erlotinib, which is an EGFR inhibitor approved by the U.S. Food and Drug Administration for the treatment of cancer, were found to be effective in vivo in promoting axonal regeneration, providing promise for treatment of brain and spinal cord injury in humans (40).
Cellular reprogramming
It was long thought that tissue/organ-specific stem/progenitor cells could give rise only to cells of the same tissue type but not to those of different tissue. In other words, they have irreversibly lost the capacity to generate cell types of other lineages in the body. However, recently several reports have demonstrated that tissue-specific stem cells may overcome their intrinsic lineage-restriction during exposure to a specific set of in vitro culture or in vivo conditions. For example, recently the Yamanaka group (41) has demonstrated that cells from mouse embryonic fibroblast culture can be induced into a pluripotent state during transduction with four genes (Oct-3/4, c-Myc, Sox2, and Klf-4) in vitro. These results were extended recently to demonstrate germline transmission of the induced pluripotent cells (42). An extreme example is the reprogramming of a somatic cell to a totipotent state by nuclear transfer cloning, where the nucleus of a somatic cell is transferred into an enucleated oocyte or the extracts of the oocyte are fused with a somatic cell (43, 44). Although in mammals neither transdifferentiation nor dedifferentiation has yet been identified as a naturally occurring process (except in certain disease states), the discovery of cell plasticity raises the possibility of reprogramming a restricted cell’s fate. The ability to dedifferentiate or reprogram lineage-committed cells to multipotent or even pluripotent cells might overcome many obstacles associated with using ESCs and adult stem cells in clinical applications (e.g., immunocom- patibility, cell isolation and expansion, or bioethics).
To identify small molecules that induce reprogramming of lineage-committed myoblasts, a cell-based screen was designed based on the notion that lineage-reversed myoblasts would regain multipotency. Specifically, dedifferentiated myoblasts would acquire the ability to differentiate into (otherwise nonpermitted) mesenchymal cell types under conditions that typically induce differentiation of only multipotent MSCs into adipocytes, osteoblasts, or chondrocytes. A two-stage screening protocol was used in which C2 C12 myoblasts were treated initially with compounds to induce dedifferentiation; compounds were then removed, and cells were assayed for their ability to undergo osteogenesis during addition of known osteogenic inducing agents. Reversine, which was a 2, 6-disubstituted purine, was found to have the desired dedifferentiation inducing activity (45). It inhibits myotube formation, and reversine-treated myoblasts can redifferentiate into osteoblasts and adipocytes during exposure to the appropriate differentiation conditions. In addition, the reprogramming effect of reversine on C2 C12 cells (as well as some other cell types) can be shown at the clonal level, which suggests its effect is inductive rather than selective. Furthermore, reversine has also been shown to induce reprogramming of primary mouse and human fibroblasts to a multipotent state, which can be redifferentiated to functional myoblasts and myotubes under myogenic conditions, which suggests that reversine’s reprogramming mechanism might be general to different cell types (46).
Affinity chromatography experiments revealed that nonmuscle myosin II heavy chain (NMMII) and MEK1 are cellular targets of reversine. Mechanistic analysis demonstrated that reversine acts by inhibiting both NMMII and MEK1. Inhibition of NMMII induces G2/M phase accumulation/staging and cytoskeletal remodeling, whereas MEK1 inhibition serves to modulate signaling, including acetylation of histone H3. Additionally, PI3K signaling was found to be essential to reversine activity (47).
Modulators of developmental pathways and epigenetic modifiers
Developmental signaling pathways, such as Wnt, Hh, TGF/ BMP, and Notch, control embryonic patterning and cell behavior during development and play important roles in regulating tissue homeostasis and regeneration in adulthood. The known chemical modulators of these developmental pathways have been used widely, including GSK3P inhibitors as agonists of a canonical Wnt pathway, fumagillin as an antagonist of a noncanonical Wnt pathway, purmorphamine as an agonist and cyclopamine as an antagonist of an Hh pathway, DAPT as an antagonist of a Notch pathway, and SB431542 as an inhibitor of TGFp/Activin signaling (18, 34, 48-50).
Although activation of Wnt, Hh, and Notch pathways is involved in various regenerative processes, their aberrant activities are also associated with cancer induction. Strategies to control the activity of these pathways for regeneration while avoiding tumor induction are highly attractive. One possible approach would be enhancing the desired pathway activity by a synergistic agonist, which would be effective only at where it is needed (i.e., the pathway activity is inadequate), rather than using a general pathway activator, which ectopically activates the signaling. To identify novel compounds and pathways that interact with the canonical Wnt/β-catenin signaling pathway, a reporter-based screen was carried out recently for molecules that synergistically activate signaling in the presence of Wnt3a. A 2,6,9-trisubstituted purine compound, QS11, was identified to synergize with canonical Wnt ligand both in vitro and in vivo (51). Affinity chromatography identified ARF-GAP as a target of QS11. Additional functional studies have confirmed that QS11 inhibits the ARF-GAP function, and as a consequence, it modulates ARF activity and β-catenin localization. Because ARFs play important roles in endocytosis, this study established another link between the endocytosis pathway and Wnt signaling, and it provides a useful chemical tool to modulate the Wnt pathway and explore novel functions of ARF-GAPs in cell culture and whole organisms.
Epigenetic modifications are central processes in stem cell differentiation and reprogramming that can control specific and heritable gene expression pattern in cells without altering the DNA sequence. Therefore, molecules directly regulating epigenetic machineries and changing the epigenetic status of cells should affect cell fate. Major epigenetic modifications include DNA methylation and histone modifications (acetylation, phosphorylation, methylation, and ubiquitination). 5-azacytidine and its analogs are widely used DNA demethylation agents, and they have been shown to increase cellular plasticity or induce differentiation of certain stem/progenitor cells (52, 53). HDAC inhibitors (e.g., TSA and VPA) have also played essential roles in studying histone acetylation and have been developed for the treatment of cancers. Like all signaling/epigenetic modulators, their effects are context-dependent; they have been shown to enhance self-renewal of HSCs, induce neuronal differentiation of NSCs, or promote myogenesis of muscle cells (54-56). In addition to these two widely used epigenetic modifiers, small-molecule inhibitors for protein arginine methyltrans- ferases (PRMTs) (41), histone methyltransferases (HMTs, e.g., Suv39H1) (42) and histone demethylases (e.g., LSD1) (43) have been identified recently via various approaches. With ongoing efforts of generating additional, more precise epigenetic chemical probes, these modifiers will no doubt contribute substantially to epigenetic control and stem cell regulation.
Conclusion
Stem cell research provides tremendous opportunities for understanding human development, regeneration, and diseases, and it offers great promise for developing cell-based or drug therapies to treat devastating diseases and injuries. Small molecules have proven to be useful probes of stem cell biology, from stimulation of homogenous self-renewal or differentiation conditions for stem cells that are essential for future cell-based therapy, to facilitation of drug development for controlling endogenous regeneration and treating cancers. However, many challenges remain, including the design of better chemical libraries and screening strategies to identify systematically small molecules that regulate the desired cellular process; developing more efficient methods to understand the underlying mechanism; and translating in vitro discoveries into approaches for in vivo regeneration of desired tissues/organs by small-molecule therapeutics. Nonetheless, it is clear that identification of additional small molecules that control stem cell fate will significantly facilitate studies of stem cell biology and contribute to the development of regenerative medicine.
References
1. Services. D.o.H.a.H. Regenerative Medicine 2006. http://stemcells.nih.gov/info/scireport/2006report.htm.
2. Pardal R, Clarke MF, Morrison SJ. Applying the principles of stem-cell biology to cancer. Nat. Rev. Cancer 2003; 3:895-902.
3. Shachaf CM, et al. MYC inactivation uncovers pluripotent differentiation and tumour dormancy in hepatocellular cancer. Nature 2004; 431:1112-1117.
4. Lapidot T, et al. A cell initiating human acute myeloid leukaemia after transplantation into SCID mice. Nature 1994; 367:645-648.
5. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat. Med. 1997; 3:730-737.
6. Al-Hajj M, et al. Prospective identification of tumorigenic breast cancer cells. Proc. Natl. Acad. Sci. U.S.A. 2003; 100:3983-3988.
7. Singh SK, et al. Identification of human brain tumour initiating cells. Nature 2004; 432:396-401.
8. Bohacek RS, McMartin C, Guida WC. The art and practice of structure-based drug design: a molecular modeling perspective. Med. Res. Rev. 1996; 16:3-50.
9. Sills MA, et al. Comparison of assay technologies for a tyrosine kinase assay generates different results in high throughput screening. J. Biomol. Screen. 2002; 7:191-214.
10. Evans BE, et al. Methods for drug discovery: development of potent, selective, orally effective cholecystokinin antagonists. J. Med. Chem. 1988; 31:2235-2246.
11. Nicolaou K, et al. Combinatorial synthesis of novel and potent inhibitors of NADH: ubiquinone oxidoreductase. Chem. Biol. 2000; 7:979-992.
12. DeSimone RW, et al. Privileged structures: applications in drug discovery. Comb. Chem. High Throughput Screen. 2004; 7:473-494.
13. Ding S, et al. A combinatorial scaffold approach toward kinase-directed heterocycle libraries. J. Am. Chem. Soc. 2002; 124:1594-1596.
14. Carpenter AE, et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol. 2006; 7:R100.
15. Carpenter AE, Sabatini DM. Systematic genome-wide screens of gene function. Nat. Rev. Genet. 2004; 5:11-22.
16. Molofsky AV, Pardal R, Morrison SJ. Diverse mechanisms regulate stem cell self-renewal. Curr. Opin. Cell. Biol. 2004; 16:700-707.
17. Ying QL, et al. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 2003; 115:281-292.
18. Sato N, et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat. Med. 2004; 10:55-63.
19. Androutsellis-Theotokis A, et al. Notch signalling regulates stem cell numbers in vitro and in vivo. Nature 2006; 442:823-826.
20. Singla DK, et al. wnt3a but not wnt11 supports self-renewal of embryonic stem cells. Biochem. Biophys. Res. Commun. 2006; 345:789-795.
21. Ito K, et al. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat. Med. 2006; 12:446-451.
22. Morrison SJ, Kimble J. Asymmetric and symmetric stem-cell divisions in development and cancer. Nature 2006; 441:1068-1074.
23. Lee HS, et al. Clonal expansion of adult rat hepatic stem cell lines by suppression of asymmetric cell kinetics (SACK). Biotechnol. Bioeng. 2003; 83:760-771.
24. Watanabe K, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 2007; 25:681-686.
25. Moretti A, et al. Multipotent embryonic isl1+ progenitor cells lead to cardiac, smooth muscle, and endothelial cell diversification. Cell 2006; 127:1151-1165.
26. Qyang Y, et al. The renewal and differentiation of isl+1 cardiovascular progenitors are controlled by a wnt/^-catenin pathway. Cell Stem. Cell. 2007. In press.
27. Wobus AM, et al. Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes. J. Mol. Cell. Cardiol. 1997; 29:1525- 1539.
28. Shi Y, et al. Inducing embryonic stem cells to differentiate into pancreatic beta cells by a novel three-step approach with activin A and all-trans retinoic acid. Stem Cells 2005; 23:656-662.
29. Seamon KB, Daly JW. Forskolin: a unique diterpene activator of cyclic AMP-generating systems. J. Cyclic Nucleotide Res. 1981; 7:201-224.
30. Warashina M, et al. A synthetic small molecule that induces neuronal differentiation of adult hippocampal neural progenitor cells. Angew Chem. Int. Ed. 2006; 45:591-593.
31. Wichterle H, et al. Directed differentiation of embryonic stem cells into motor neurons. Cell 2002; 110:385-397.
32. Grigoriadis AE, Heersche JN, Aubin JE. Differentiation of muscle, fat, cartilage, and bone from progenitor cells present in a bone-derived clonal cell population: effect of dexamethasone. J. Cell Biol. 1988; 106:2139-2151.
33. Jaiswal N, et al. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J. Cell Biochem. 1997; 64:295-312.
34. Wu X, et al. Purmorphamine induces osteogenesis by activation of the hedgehog signaling pathway. Chem. Biol. 2004; 11:1229-1238.
35. Sinha S, Chen JK. Purmorphamine activates the Hedgehog pathway by targeting Smoothened. Nat. Chem. Biol. 2006; 2:29-30.
36. Studzinski GP, Harrison LE. Differentiation-related changes in the cell cycle traverse. Int. Rev. Cytol. 1999; 189:1-58.
37. Engel FB, et al. p38 MAP kinase inhibition enables proliferation of adult mammalian cardiomyocytes. Genes Dev. 2005; 19:1175-1187.
38. Tseng AS, Engel FB, Keating MT. The GSK-3 inhibitor BIO promotes proliferation in mammalian cardiomyocytes. Chem. Biol. 2006; 13:957-963.
39. Rosania GR, et al. Myoseverin, a microtubule-binding molecule with novel cellular effects. Nat. Biotechnol. 2000; 18:304-308.
40. Koprivica V, et al. EGFR activation mediates inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans. Science 2005; 310:106-110.
41. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:663-676.
42. Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluripotent stem cells. Nature 2007; 448:313-317.
43. Hakelien AM, et al. Reprogramming fibroblasts to express T-cell functions using cell extracts. Nat. Biotechnol. 2002; 20:460-466.
44. Lanza RP, et al. Generation of histocompatible tissues using nuclear transplantation. Nat. Biotechnol. 2002; 20:689-696.
45. Chen S, et al. Dedifferentiation of lineage-committed cells by a small molecule. J. Am. Chem. Soc. 2004; 126:410-411.
46. Anastasia L, et al. Reversine-treated fibroblasts acquire myogenic competence in vitro and in regenerating skeletal muscle. Cell Death Differ. 2006.
47. Chen S, Takanashi S, Zhang Q, Xiong W, Peters EC, Schultz PG. Reversine induces cellular reprogramming of lineage-committed mammalian cells. Proc. Natl. Acad. Sci. U.S.A. 2007.
48. Dovey HF, et al. Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J. Neurochem. 2001; 76:173- 181.
49. Watabe T, et al. TGF-beta receptor kinase inhibitor enhances growth and integrity of embryonic stem cell-derived endothelial cells. J. Cell. Biol. 2003; 163:1303-1311.
50. Zhang Y, et al. A chemical and genetic approach to the mode of action of fumagillin. Chem. Biol. 2006; 13:1001-1009.
51. Zhang Q, et al. Small-molecule synergist of the Wnt/beta-catenin signaling pathway. Proc. Natl. Acad. Sci. U.S.A. 2007; 104:7444-7448.
52. Choi SC, et al. 5-azacytidine induces cardiac differentiation of P19 embryonic stem cells. Exp. Mol. Med. 2004; 36:515-523.
53. Tsuji-Takayama K, et al. Demethylating agent, 5-azacytidine, reverses differentiation of embryonic stem cells. Biochem. Biophys. Res. Commun. 2004; 323:86-90.
54. Hsieh J, et al. Histone deacetylase inhibition-mediated neuronal differentiation of multipotent adult neural progenitor cells. Proc. Natl. Acad. Sci. U.S.A. 2004; 101:16659-16664.
55. Iezzi S, et al. Deacetylase inhibitors increase muscle cell size by promoting myoblast recruitment and fusion through induction of follistatin. Dev. Cell 2004; 6:673-684.
56. De Felice L, et al. Histone deacetylase inhibitor valproic acid enhances the cytokine-induced expansion of human hematopoietic stem cells. Cancer Res. 2005; 65:1505-1513.