Exploring stem cell biology with small molecules

Abstract

Stem cells hold promise for the treatment of a number of diseases. Small molecules serve as useful chemical tools to control stem cell fate and may ultimately contribute to development of effective medicines for tissue repair and regeneration.

Shuibing Chen

Shuibing Chen was born in Anshan, China. She received her BS (1999) and MS (2002) in chemistry from Tsinghua University, Beijing, China, where she conducted research on the structural effect of protein phosphorylation. She is currently a PhD candidate working on regulating stem cell fate with chemical tools under the supervision of Professor Peter G. Schultz in the Department of Chemistry at the Scripps Research Institute.

Simon Hilcove

Simon Hilcove (1979, Pietermaritzberg, South Africa) was raised in Canada and received his BS at Arizona State University. His undergraduate research with Dr. G. Ghirlanda focused on protein design. He is now a graduate student at the Scripps Research Institute in the laboratory of Professor Sheng Ding where his research focuses on cardiomyocyte differentiation of embryonic stem cells.

Sheng Ding

Sheng Ding received his BS from Caltech (1999), and PhD in chemistry from the Scripps Research Institute (2003). He then joined the faculty of the Chemistry and Cell Biology Departments at Scripps as an Assistant Professor in 2003. His research interest is to develop and integrate chemical and functional genomic tools to study stem cell biology and regeneration, focusing on identification and characterization of small molecules and genes that can control self-renewal and differentiations of embryonic stem cells and various adult stem cells (e.g. neural stem cells and mesenchymal stem cells), as well as cellular plasticity and dedifferentiation of lineage-restricted somatic cells.

Introduction

A stem cell is an extraordinary type of cell that has the ability to self-renew for long periods of time and to differentiate into specialized cells under appropriate physiological or experimental conditions.¹ Traditionally, stem cells are classified as either embryonic or adult (tissue-specific) stem cells. Embryonic stem cells (ESCs) are derived from the inner cell mass of the blastocyst stage embryo. They possess an unlimited capacity for self-renewal and have the potential to develop into any cell type found in the three primary germ layers of the embryo (endoderm, mesoderm and ectoderm), as well as germ cells and extraembryonic cells.^2–4 In contrast, adult stem cells are found in differentiated tissues, have limited self-renewal capability, and generally can only differentiate into specialized (mature) cell types of the tissue in which they reside.

Recent advances in stem cell biology may provide new approaches for the treatment of a number of diseases as well as tissue/organ injuries, including cardiovascular disease, neurodegenerative disease, musculoskeletal disease, diabetes, and spinal-cord injuries.¹ These approaches could involve cell replacement therapy and/or drug treatment to stimulate the body's own regenerative capabilities by promoting survival, migration/homing, proliferation, and differentiation of endogenous stem/progenitor cells. Essential to these pursuits is the identification of renewable sources of engraftable functional cells, an improved ability to manipulate stem cell proliferation and differentiation, and a better understanding of the signaling pathways that control stem cell fate. In addition, there is a growing body of evidence supporting the notion that tumors are initiated and maintained by a small number of cancer cells with stem cell-like features: normal and cancer stem cells share similar self-renewal mechanisms; deregulation of signaling pathways involved in stem cell self-renewal is associated with oncogenesis; cancer stem cells may arise from normal stem cells or through transformations of progenitor cells.⁵ A better understanding of stem cell biology may also contribute to development of improved therapies for cancers.

Stem cell fate is determined by both intrinsic regulators and the extra-cellular environment (niche), and their expansion and differentiation ex vivo are generally controlled by growing them in a specific configuration (monolayer or three-dimensional culture) with “cocktails” of growth factors and signaling molecules, as well as genetic manipulations. However, most of these conditions are either incompletely defined, or non-specific in regulating the desired cellular process. Undefined media often result in inconsistency in cell culture and/or heterogeneous populations of cells which would not be useful for cell-based therapy and would complicate the biological study of a particular cellular process. More efficient and selective methods to control the fate of stem cells to produce homogenous populations of particular cell types will be essential to the therapeutic use of stem cells and will facilitate studies of the molecular mechanisms of development. Cell-based phenotypic and, more recently, pathway-specific screens of synthetic small molecules and natural products have provided useful chemical tools to modulate and study complex cellular processes.^6–10 Cell permeable small molecules (Fig. 1) such as L-ascorbic acid, 5-azacytidine (5-aza-C), dexamethasone, all-trans retinoic acid (RA), and trichostatin A (TSA) have proven extremely useful in modulating stem cell fate. In this article, we focus on recent developments in the use of small molecules to control self-renewal, differentiation, dedifferentiation and proliferation in embryonic and adult stem cells, as well differentiated/lineage-restricted cells. In addition, we also briefly review the signaling pathways regulating stem cell fate.

Fig. 1 Structures of small molecules that regulate stem cell fate.

Self-renewal

Self-renewal is the process by which a stem cell divides to generate one (asymmetric division) or two (symmetric division) daughter stem cells without the loss of developmental potential.¹¹ The ability of stem cells to self-renew is critical to the development and maintenance of adult tissues. Pluripotent ESCs and multipotent hematopoietic stem cells (HSCs) provide useful model systems to analyze the molecular mechanisms of stem cell self-renewal.

ESC self-renewal

Mouse ESCs (mESCs) are conventionally maintained on feeder cells and/or mixtures of exogenous factors (e.g. serum). Studies have found that the self-renewal of mESCs primarily depends on two key signaling molecules: leukemia inhibitory factor (LIF)/interleukin 6 (IL6) family members and BMP. LIF activates Stat (signal transduction and activation of transcription) signaling via a membrane-bound gp130-LIFR (LIF receptor) complex, thereby promoting self-renewal and inhibiting differentiation of mESCs by a Myc-dependent mechanism. BMP induces expression of Id (inhibitor of differentiation) genes via Smad signaling and inhibits mESC differentiation to neuroectoderm. The combination of BMP and LIF can sustain mESCs self-renewal in the absence of serum and feeder cells.¹² LIF stimulation also activates the mitogen-induced extracellular kinase (MEK)/extracellular signal-related kinase (ERK) signaling, which was shown to be a negative regulatory pathway for self-renewal of mESCs. In contrast, BMP signaling was shown to inhibit both ERK and p38 mitogen-active protein kinase (MAPK) pathways.¹³ Consistent with these observations, PD98059, a chemical MEK inhibitor, was shown to enhance (but is insufficient to support) the self-renewal of mESCs by inhibiting the MEK/ERK signaling pathway.¹⁴ SB203580, a p38 inhibitor, was also shown to have a positive effect in maintaining mESCs and support the derivation of ES cell lines from Alk3^−/− embryos.¹⁴ In addition, cell death and differentiation are linked processes and it has been shown that upon the withdrawal of LIF, some mESCs undergo apoptosis with a concomitant activation of p38 MAPK. Interestingly, the apoptosis of mESCs induced by LIF withdrawal can also be prevented by the p38 inhibitors, PD 169316 and SB203580.¹⁵

6-Bromoindirubin-3′-oxime (BIO), a natural product derived from mollusk Tyrian purple, has been recently shown to maintain mESCs in the pluripotent state without LIF. mESCs expanded under BIO treatment can generate teratomas consisting of all three germ layer-derived tissues, including neuroepithelium (ectoderm), cartilage (mesoderm) and ciliated epithelium (endoderm). In addition, BIO treated mESCs can contribute to chimeric animals after being injected into blastocysts. BIO is proposed to function by inhibiting GSK3β, resulting in activation of a canonical Wnt signaling pathway. To confirm this mechanism, recombinant Wnt3a protein was shown to have a similar effect as BIO on mESC self-renewal.¹⁶ One of the downstream substrates of GSK3β in the mESC self-renewal process is suggested to be Myc, which is also regulated by Stat3 at the transcriptional level. In undifferentiated mESCs, Myc mRNAs are expressed in elevated levels and Myc proteins are stabilized in the unphosphorylated form. Following LIF withdrawal, Myc mRNA expression is down-regulated and Myc proteins become phosphorylated on threonine 58 (T58) by GSK3β (which is dependent on the prior phosphorylation of Ser 62 likely mediated through the action of ERK), leading to their degradation. BIO seems to play a role in suppressing GSK3β-mediated Myc-T58-phosphorylation, sustains Myc stability and maintains the pluripotency of mESCs.¹⁷

There are significant differences in cell culture requirements for the maintenance of mESCs and hESCs. For example, LIF has no obvious effect in supporting hESC self-renewal and BMP signaling works as a differentiation inducer in the current culture system of hESCs.^18,19 Interestingly, BIO-mediated ESC self-renewal is also shown in hESCs. After 7 days treatment with BIO, hESCs maintain Oct3/4 expression and compact colony morphology. hESCs treated by BIO seem to preserve their pluripotency, as they can form differentiating EBs which express markers of ectoderm, mesoderm and endoderm, and also differentiate into neurons by specific culture techniques. However, such self-renewal studies have only been conducted over a brief culture period.¹⁶ Further investigation is needed to elucidate the function of Wnt signaling in hESCs. Recently, using a feeder cell dependent mES cell line and an imaging-based assay that combines expression (Oct4, a pluripotency marker) and morphological (undifferentiated ESCs grow as compact colonies) analysis, we identified a synthetic small molecule that can functionally maintain the self-renewal of mESCs without feeder cells and LIF in multiple passages. We also showed that the identified compound has a positive effect in promoting self-renewal of hESCs. Interestingly, this compound activates neither the Wnt pathway nor the JAK-STAT pathway and therefore may provide new opportunities for characterizing fundamental mechanisms of ESC self-renewal (Chen, et al., unpublished data).

HSC self-renewal

HSCs are circulating stem cells in the blood and are typically isolated from the bone marrow. They can generate all blood-lineage precursor and mature cell types and have long been used for treating a variety of malignant and non-malignant hematological diseases (e.g. cancers and autoimmune disorders), and have been shown in some conditions to contribute to the regeneration of skeletal muscles, liver cells and endothelial cells.^20,21 Though HSCs self-renew in vivo throughout life, their ex vivo expansion is still challenging. Recently, a chemical inhibitor of histone deacetylase (HDAC), valproic acid (VPA), was reported to increase chromatin accessibility, which resulted in the concomitant enhancement of the cytokine effect on the maintenance and expansion of a primitive HSC population.²² This may have important therapeutic consequences and expand the response to chemotherapeutic agents in stem cell transplantation.

Differentiation

Differentiation is a process involving unspecialized cells progressing to become specialized cells with restricted developmental potentials. Under appropriate conditions in cell culture, stem cells can differentiate spontaneously. For example, the most commonly used method for inducing differentiation of mESCs involves growing them in suspension (in the presence of serum and absence of supplemented LIF) to form aggregates called embryoid bodies (EBs) which begin to differentiate spontaneously into various cell types, including hematopoietic, endothelial, neuronal and cardiac muscle cells. However, such in vitro spontaneous differentiation of EBs involves a poorly defined, inefficient, and relatively non-selective process, and therefore leads to heterogeneous populations of differentiated and undifferentiated cells. Consequently, dissecting stem cell signaling pathways and identifying critical factors that are involved in tissue specification are essential for developments of stem cell therapy and related small molecule therapeutics. A number of small molecules have been identified that modulate specific differentiation pathways of embryonic or adult stem cells.

Neural and neuronal differentiations

RA is a widely used small molecule for neural and neuronal differentiations of ESCs and neural cells. It was recently demonstrated that subtype-specific neurons can be generated from mouse and human ESCs in a stepwise fashion. For example, to generate motor neurons, mESCs were first neuralized through EB formation with concomitant RA treatment. The generated neural cells were further caudalized by RA, followed by treatment with a specific small molecule agonist (Hh-Ag1.3) of Sonic hedgehog (Shh) signaling to ventralize the caudalized neural cells to become the desired motor neurons.²³ This experiment suggests that multiple sequential and/or combination of signals may be required to generate a terminally differentiated, subtype-specific cell type.

TWS119, a synthetic disubstituted pyrrolopyrimidine, was recently identified from a cell-based screen as a potent inducer of neuronal differentiation in pluripotent mESCs and P19 murine embryonal carcinoma cells (mECCs).²⁴ A panel of affinity matrices, prepared from representative TWS analogs, were used to pull-down target proteins from P19 cell extracts. Proteins specifically bound to all positive resins derived from active molecules but not to the negative resins derived from inactive molecules were considered to be the putative targets of TWS119. Consequently, GSK3 was identified as one target of TWS119, and confirmed by biochemical and cellular assays (e.g. surface plasmon resonance, kinase inhibition assay, Western blot, and reporter assay, etc.). This target identification may provide yet another link between neuronal differentiation and the Wnt signaling pathway. Additional studies also indicated that TWS119 (like BIO) is not entirely specific against GSK. Alternatively, TWS119 might promote neuronal differentiation of mESCs via novel mechanisms other than the canonical Wnt signaling pathway. Such mechanisms might include the inhibition of one or more kinases that were not apparent in the affinity experiments (possibly due to low abundance or other factors), or other proteins involved in controlling stem cell fate.

Cardiomyogenic differentiation of ESCs

The mammalian adult heart is mainly composed of post-mitotic and terminally differentiated cells. In addition to the approaches using cardiac cells (e.g. adult cardiomyocytes and cardiac stem cells, which have limited availability) and non-cardiac progenitor cells (via transdifferentiation to overcome their lineage restriction, which is still very inefficient) to repair injured heart, ESCs represent an alternative unlimited source of functional cardiomyocytes. Using mESCs stably transfected with the cardiac muscle specific α-myosin heavy chain (αMHC) promoter-driven enhanced green fluorescence protein (EGFP) as a reporter, Takahashi et al. screened 880 known drugs in monolayer culture and found that ascorbic acid (vitamin C) can significantly enhance spontaneous cardiac differentiation of mESCs.²⁵ Interestingly, other antioxidants such as N-acetylcysteine or vitamin E do not have a similar effect, suggesting that the cardiomyogenesis inducing activity of ascorbic acid may be independent of its antioxidative property. Concurrent with this work, we screened large combinatorial chemical libraries using P19 cells that were stably transfected with the cardiac muscle specific atrial natriuretic factor (ANF) promoter-driven luciferase reporter, and found a series of diaminopyrimidine compounds, named cardiogenol A–D, that can efficiently and selectively induce P19 and mESCs to differentiate into cardiomyocytes. The differentiated cells expressed multiple cardiac muscle markers, including GATA-4, Nkx2.5, MEF2, and myosin heavy chain (MHC), and formed large areas of spontaneously beating patches.²⁶

Differentiations of mesenchymal stem/progenitor cells

Mesenchymal stem cells (MSCs) are multipotent cells with significant cellular plasticity, and can differentiate into a variety of mesenchymal tissues such as osteoblasts, adipocytes and chondrocytes as well as other tissue types such as neuronal and skeletal muscle cells under specific differentiation conditions.^27,28 A number of small molecules have been found that can be used to control the differentiation of mesenchymal stem/progenitor cells for a variety of applications. For example, 5-aza-C (a DNA demethylation chemical) can induce C3H10T1/2 cells (a mouse mesenchymal progenitor cell line) to differentiate into myoblasts, osteoblasts, adipocytes and chondrocytes. 5-Aza-C does not directly activate a specific differentiation program, but rather converts the cells into a competent differentiation state.²⁹ Dexamethasone (a glucocorticoid receptor agonist), ascorbic acid, β-glycerophosphate, isobutylmethylxanthine (IBMX, a nonspecific phosphodiesterase inhibitor) and peroxisome proliferator-activated receptor γ (PPARγ) agonists (such as rosiglitazone) have been widely used to modulate osteogenesis or adipogenesis of MSCs under carefully defined conditions.³⁰ Interestingly, treatment with a JAK inhibitor (WHI-P131), followed by trophic factor induction, was recently shown to be able to convert rat MSCs into neuronal cells.²⁷ Purmorphamine, a 2,6,9-trisubstituted purine compound, was identified as a potent osteogenic differentiation inducing molecule through a high throughput chemical screen in C3H10T1/2 cells.³¹ Expression profiling of cells treated with purmorphamine in conjunction with systematic pathway analysis was used to reveal that the Hedgehog/Hh signaling pathway is the primary affected biological network and purmorphamine is a selective Hh pathway agonist, which was further confirmed by chemical epistasis using two different Hh pathway antagonists: cyclopamine that binds and inhibits Smoothened (Smo), and forskolin that activates protein kinase A (PKA), which converts Gli proteins to transcriptional repressors by phosphorylation.³² Furthermore, purmorphamine's function as an Hh pathway agonist was also confirmed by its effects of proliferating primary adult neural stem cells, as well as acting with RA in the sequential fashion to induce motor neuron differentiation of mESCs.

Dedifferentiation and nuclear reprogramming

A long-standing notion in developmental biology has been that organ/tissue-specific stem cells are restricted to differentiating into cell types of the tissue in which they reside. However, recent studies suggest that tissue-specific stem/progenitor cells may overcome their intrinsic lineage-restriction upon exposure to a specific set of signals in vitro and in vivo,^33,34 although such reprogramming may not reflect potentials that are normally exercised in vivo. 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.^35,36 The ability to dedifferentiate or reverse lineage-committed cells back to multipotent or even pluripotent cells might overcome many of the obstacles associated with using ESCs and adult stem cells. To identify small molecules that can induce dedifferentiation of C2C12 myoblasts,³⁷ an assay was designed based on the notion that lineage-reversed myoblasts should regain multipotency, the ability to differentiate into multiple mesenchymal cell lineages under conditions that typically induce differentiation of only multipotent mesenchymal stem cells into adipocytes, osteoblasts or chondrocytes. Reversine, a 2,6-disubstituted purine, was found to have the desired dedifferentiation inducing activity: it inhibits myotube formation of C2C12 myoblasts and reversine-treated myoblasts can efficiently differentiate into osteoblasts and adipocytes only upon exposure to the appropriate differentiation conditions. Importantly, the dedifferentiation effect of reversine on C2C12 cells (as well as some other cell types) can be shown at the clonal level, suggesting it is inductive rather than selective. This example is a proof-of-principle demonstration that dedifferentiation of lineage-restricted cells to a more primitive (multipotent) state by a synthetic chemical can be achieved via a rationally designed phenotypic screen of combinatorial chemical libraries, and such concepts and technologies are readily applicable to other models.

Functional proliferation of adult mature cells

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 exited the cell cycle. Their loss of the ability to regenerate (i.e. to divide and replace damaged/lost tissue) may represent a major medical problem. For example, ischemic heart disease, resulting from loss of cardiac muscle cells, is a leading cause of morbidity and mortality in the industrialized world.³⁸ Consequently, stimulation of adult cells to reenter cell cycle and proliferate may provide new therapeutic approaches for treating these types of diseases.

Cardiomyocyte proliferation

Two types of widely studied cardiomyocytes are neonatal cardiomyocytes (which are typically derived from hearts of 1-day-old newborns) and adult cardiomyocytes (which are derived from adult hearts and have terminally differentiated and exited the cell cycle). Because mammalian cardiomyocytes still proliferate during fetal development, the neonatal cardiomyocytes have some limited proliferative ability. Several growth factors can promote DNA synthesis and proliferation in neonatal cardiomyocytes, including FGF1, IL-1β, and NRG-1-β1.^39,40 Shortly after birth, the cell cycle-perpetuating factors (e.g. cyclin A and cdk2) are down-regulated in the cardiomyocytes and they lose their proliferative capacity.³⁹ p38 MAPK was identified as a key negative regulator of mammalian cardiomyocyte proliferation through regulating genes required for mitosis (including cyclin A and cyclin B). Recently, it was reported that a p38 inhibitor, SB203580, increased the growth-factor-induced DNA synthesis (measured by BrdU incorporation) and mitosis (histone 3 phosphorylation) in both neonatal and adult cardiomyocytes. This proliferation in adult cardiomyocytes was also observed to be associated with transient dedifferentiation of the contractile apparatus.⁴¹

β-cell proliferation

One potential “cure” for type 1 diabetes, which results from an autoimmune destruction of the insulin-secreting β cells in the pancreatic islets, is the transplantation of functional glucose-responsive islets with simultaneous prevention of their immune destruction. The major obstacles to this approach are the shortage of engraftable donor tissues and lifelong immune suppression. Alternatively, stimulation of endogenous β cell regeneration in vivo by small molecule therapeutics may represent a new approach for the treatment of type 1 diabetes. In addition, an efficient approach for β cell proliferation in vitro may also provide an expanded source of (and potentially immunocompatible) functional β cells for islet transplantation. Cytokines, such as insulin-like growth factors, hepatocyte growth factors and pituitary adenylate cyclase-activating polypeptide, are capable of increasing β-cell mass in an experimental model of type 2 diabetes.^42,43 Exendin 4 (EX-4), a long-lasting analog of the intestinal hormone glucagon-like peptide 1 (GLP-1), can stimulate the proliferation of β-cells when given to type 2 diabetic mice.⁴⁴ However, none of these proliferating cells retain insulin and c-peptide expression. Recent high throughput chemical screening of proliferation in primary human β-cells and follow-up characterization studies in our laboratory have suggested that the functional proliferation of human β-cells (i.e. proliferation while retaining β-cell functions) may be possible by manipulating a developmental signaling pathway via small molecules, and such studies may open up an alternative avenue toward development of new therapies for type 1 diabetes (Chen, et al., unpublished data).

Developmental signaling pathways

Fundamental developmental signaling pathways (e.g. Wnt, Hh, BMP and Notch etc.), which control embryonic patterning and cell behaviors, play important roles in stem cell regulation. Deregulation of these pathways in either the embryonic or adult stage may result in diseases, such as cancer and degenerative disease. For example, abnormal activation of the Hh pathway has been implicated in progression of a number of cancers, including basal cell carcinoma, small cell lung cancer, medulloblastoma, digestive tract tumours and prostate cancers.⁴⁵ As described above, some of the stem cell fate regulators are pathway-specific modulators, such as purmorphamine and cyclopamine—Hh pathway agonist and antagonist,³² respectively; BIO and sulindac—Wnt pathway agonist and antagonist, respectively; and NS-phenyl-glycine-t-butyl ester (DAPT)—Notch pathway antagonist.^46,47 Cell-based pathway-specific screens have been used to identify small molecule regulators of these developmental pathways. For example, a series of Hh pathway specific agonists and antagonists have been identified through screens of synthetic compounds using 10T1/2 cells stably transfected with a luciferase reporter downstream of multimerized Gli binding sites and a minimal promoter.³² We recently carried out Wnt pathway specific screens of chemical libraries using a TOPflash reporter assay, and have identified a 2-amino-4,6-disubstituted pyrimidine compound that activates Wnt signaling in a dose-dependent manner. This compound does not inhibit GSK-3β, a major inhibitory component in the pathway, but its activity can be blocked by a dominant negative TCF4, suggesting that this compound functions upstream of the known TCF factors on the canonical Wnt signaling pathway. Importantly, this compound appears to mimic the effects of Wnt ligand in a Xenopus model, suggesting it may be a useful tool to study physiological processes that involve Wnt signaling.⁴⁸

Epigenetic modifications

Epigenetic modification can regulate lineage specification by imposing a specific and heritable pattern of gene expression on the progeny of cells, without altering the DNA sequence. Major epigenetic modifications include DNA methylation, histone acetylation and methylation, and histone phosphorylation. DNA methylation, occurring progressively during differentiation of stem cells, typically induces gene silencing. DNA demethylation has been associated with increased cellular plasticity of lineage-committed cells. For example, after the transplantation of mammalian nuclei into Xenopus oocytes, DNA demethylation at the Oct-4 promoter preceded gene reprogramming and Oct4 transcription.⁴⁹ Another example is the demethylation chemical, 5-AzaC, which can reverse the differentiation of ESCs. After 3 days treatment of 5-AzaC, the differentiated cells from day 7 and day 11 EBs regained the stem cell-like colony morphology and expression of ESC specific markers;⁵⁰ and the promoter region of the H19 gene, which is methylated during EB differentiation, was found to be demethylated.

Histone acetylation is another widely studied epigenetic modification. In specific cellular contexts, some HDAC inhibitors can induce specific differentiation of stem cells. For example, VPA induced neuronal differentiation of multipotent adult neural progenitors, and suppressed astrocyte and oligodendrocyte differentiations even under their favored lineage-specific differentiation conditions. Mechanistic analysis revealed an up-regulation of a master neurogenic bHLH transcription factor, NeuroD, in VPA-treated cultures.⁵¹ Recently, it was shown that treatment of mESCs with TSA, an HDAC inhibitor, induced cardiac muscle differentiation associated with increased acetylation of GATA-4, a cardiac-specific transcription factor important for cardiac lineage specification.⁵²

Conclusion and perspective

Stem cell biology is a fast growing field offering new opportunities for the treatment of many devastating diseases, as well as providing new insights into the molecular mechanisms that control developmental processes. Realization of these potentials will require a better understanding of the signaling pathways that control stem cell fate and an improved ability to manipulate stem cell proliferation and differentiation. As exemplified above, synthetic molecules have been identified that regulate stem cell fate and can be used as probes of the underlying biology. However, many challenges remain, including designing better chemical libraries and screening strategies to systematically identify small molecules that regulate the desired cellular process; developing more efficient methods to understand the underlying mechanism; 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 development of regenerative medicine.

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