Stemistry: the control of stem cells in situ using chemistry.

Perspective pubs.acs.org/jmc

Stemistry: The Control of Stem Cells in Situ Using Chemistry Stephen G. Davies,*,† Peter D. Kennewell,† Angela J. Russell,*,†,‡ Peter T. Seden,† Robert Westwood,† and Graham M. Wynne† †

Department of Chemistry, University of Oxford, Chemistry Research Laboratory, Mansfield Road, Oxford, OX1 3TA, U.K. Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, OX1 3QT, U.K.

‡

ABSTRACT: A new paradigm for drug research has emerged, namely the deliberate search for molecules able to selectively affect the proliferation, differentiation, and migration of adult stem cells within the tissues in which they exist. Recently, there has been significant interest in medicinal chemistry toward the discovery and design of low molecular weight molecules that affect stem cells and thus have novel therapeutic activity. We believe that a successful agent from such a discover program would have profound effects on the treatment of many longterm degenerative disorders. Among these conditions are examples such as cardiovascular decay, neurological disorders including Alzheimer’s disease, and macular degeneration, all of which have significant unmet medical needs. This perspective will review evidence from the literature that indicates that discovery of such agents is achievable and represents a worthwhile pursuit for the skills of the medicinal chemist. • stimulation or inhibition of the adjacent cells in the niche responsible for the production of these stimulating or inhibiting mediators. The purpose of this perspective is to propose and justify a screening protocol whereby small molecules capable of causing adult stem cells to selectively proliferate and/or differentiate could be detected, identified, and optimized. The terms used in this perspective include: Self-renewal is the process whereby a stem cell divides to make at least one copy of itself. Proliferation is the process whereby a cell multiplies but does not change its cell type. Thus, the proliferation of stem cells involves the production of many more stem cells of exactly the same type. Differentiation is the process whereby a cell, in this case a stem or progenitor cell, changes into a specific cell type. Dedifferentiation or transdifferentiation are less frequently observed, but important processes whereby a differentiated cell reverts back to a progenitor cell or converts to a distinct differentiated cell type, respectively. It is proposed that using appropriate screening procedures, drugs giving organ repair by mechanisms not involving stem cells would also be detected. By applying well established techniques of medicinal chemistry, such molecules could provide a new and effective approach to the treatment of a number of currently poorly addressed degenerative conditions which affect, inter alia, the eyes, brain, heart, and liver.

1. INTRODUCTION There is emerging evidence to support the presence of residual adult stem cells in many tissues of the body which have the potential to proliferate, differentiate, and in many instances replenish or repair damage to the parent tissue.2 However, the ability to do this appears to vary considerably from tissue to tissue with, for example, stem cells in the bone marrow being very efficient at continually regenerating the cells within blood. Likewise, the cells of the intestinal lining appear to readily regenerate, and there is increasing evidence to suggest that many other tissues can activate repair mechanisms in response to injury. It is also important to note that such regeneration systems as are known to exist seem to become less efficient with age.3 The prospect of developing agents capable of targeting these (in many cases apparently latent) repair processes in situ, i.e., within the tissues in which they exist, is an emerging opportunity in medicine which may overcome some of the potential concerns in stem cell transplantation therapies such as immune rejection or failure to engraft and functionally integrate into existing tissue. We have named this process “stemistry” and believe that a successful agent from such a discover program would have profound effects on the treatment of many long-term degenerative disorders.1 Through illustrative examples, this perspective will highlight for the medicinal chemist recent examples of the development of agents acting through one or more of mechanisms which could include: • direct targeting of the stem cells themselves; • interference with the functioning of the endogenous high molecular weight mediators found in the microenvironment of the target niche; © XXXX American Chemical Society

Received: June 2, 2014

A

DOI: 10.1021/jm500838d J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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few examples of in vivo active molecules presumably reflect the increased complexity and the limited current state of fundamental knowledge of these systems. The development of active molecules from such studies via the sequence, in vitro−in(or ex)vivo−whole animal clinical studies and the many problems associated with this will be discussed. The perspective will not discuss cell transplantation therapies or the development of high molecular weight mediators which acted as a forerunner to this medicinal chemistry paradigm and which are extensively reviewed elsewhere.9 Also outside the scope of this perspective is any discussion of the role of cancer stem cells and their targeting as potential anticancer treatments. As this is a perspective and not a review, only a limited, noncomprehensive range of published studies in this area will be included to illustrate the points being made. 1.1. Introduction to Stem Cells in Nature. Stem cells possess two critical properties: an ability to repeatedly selfreplicate (self-renew) through cell division while maintaining an undifferentiated state and the capacity to differentiate into specialized cell types. Stem cells are conveniently classified according to their ability to differentiate into a variety of cell types: Totipotent stem cells are only found in the fertilized egg and its first few cell divisions. They are capable of producing all of the more than 200 different cell lines found in the human body including the placenta and extra-embryonic tissue. Pluripotent stem cells can make all of the cell lines in embryonic and adult tissue, with the exception of the placenta and extra-embryonic tissue. Multipotent stem cells can only make a number of closely related cells, usually concerning a particular organ or tissue. Oligopotent stem cells produce lymphoid or myeloid cell types. Unipotent stem cells can make only one cell type but still reproduce themselves, an ability which differentiates them from nonstem cells. Pluripotent embryonic stem cells are derived from the epiblast tissue of the inner cell mass of the very early stage embryo (approximately 4−5 days of age in the human) and consist of 50−150 cells. This stage of development of the embryo is known as the blastocyst, and the inner cell mass eventually forms the embryo proper. Nowadays, most human embryonic stem cells (ESCs) are derived from fertilized eggs which have been donated for research purposes, but historically, they could not be isolated without bringing about the death of the embryo and this has made their use unacceptable to a significant number of religious and cultural groups. Adult, or somatic, stem cells are found in both children and adults and occur throughout the body. Multipotent adult stem cells are rare but are found in bone marrow and umbilical cord blood and bone marrow derived cells. To date, these have been evaluated for therapeutic use in areas such as spinal cord injury, liver cirrhosis, chronic limb ischemia, and end stage heart failure. More common are tissue specific multipotent stem cells which are able to produce the cells of the tissue or organ in which they are found. The use of bone marrow transplants to treat leukemia and related blood/bone cancers is well established, and this will be discussed later. A progenitor cell, like a stem cell, has a tendency to differentiate into a specific type of cell but is already more specific than a stem cell and is pushed to differentiate into its “target” cell (Figure 1). The most important difference between stem cells and progenitor cells is that stem cells can in principle

Particular attention will be paid to testing protocols involving phenotypic screening and model organisms. These enable the discovery of active molecules both from lead structures with defined pharmacology and by high-throughput screening of large compound libraries. The modification of these active compounds by conventional medicinal chemistry techniques to maximize the potency and selectivity of candidate compounds will be discussed and, where appropriate, the leading “proof-ofprinciple” compounds presented. While there have been many examples of drug approvals from phenotypic screening campaigns, there can be challenges in translating candidate compounds to the clinic where the molecular target, or off-target effects, may be unknown. It is clear from the multiple studies described that the detailed investigation of the mechanisms of action of these compounds will produce valuable information on the critical signaling pathways in the cells and present further targets for drug discovery; examples of these will be discussed. More detailed investigations of the pharmacological actions of significant compounds in a wide range of cell and tissue types may be used to give a detailed picture of their overall biological activity, tissue specificity, and safety profile before the undertaking of clinical studies. Thus, through the use of key examples, this perspective will pay particular attention to: (a) documenting what is already known about molecules acting on these cell systems; (b) reviewing the state of the art for initial screening protocols which have the potential to be used to discover active compounds by both traditional and high-throughput assays; (c) describing the more detailed studies necessary on key compounds before human use could be envisaged; (d) describing such compounds which have been used in the clinic. The concept of stimulating stem cells in situ is an emerging approach in regenerative medicine, and a few examples of candidate drugs operating through this mechanism have been reported and reviewed.4,5 Recently Hoffman and Metternich have used the term “molecular organ repair”6 to describe the processes whereby small molecules are able to trigger cellular self-renewal mechanisms or cell-type specific activation of somatic stem cells. Further, Poss has discussed tissue regeneration and pointed out that many mammalian tissues, including cardiac muscle, spinal cord, and the major appendages, appear to have very little regenerative capacity.7 He also pointed out that stem cells are not always involved even in the well-established regenerative processes. Indeed, the example par excellence of limb regeneration, namely the salamander, appears to use predominantly a process of dedifferentiation rather than stem cell differentiation. Further, in the mammalian heart, multiple potential candidate cell types have been identified. However, there appears to be more promise in targeting resident progenitor cells of developmental origin which contribute multiple cell types to the developing heart for cardiac regeneration as opposed to the rare adult-only resident cardiac “stem cells”.8 While the objective of these studies is obviously to find in vivo active molecules and several pioneering examples have been described in recent years, the majority of the work disclosed to date has come from in vitro studies. Nonetheless, these systems represent useful in vitro models and selected examples will be discussed in this context. The comparatively B



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2. IN VITRO SCREENING SYSTEMS 2.1. Overview of Screening Procedures. High-throughput screening (HTS) of large libraries of compounds is a wellrecognized drug discovery technique which has led to significant numbers of drug candidates even if it has yet to deliver the enhanced growth in clinically useful molecules. While traditionally HTS focuses on recombinant enzyme or receptor screens, recently there has been a move toward phenotypic-based and high-content systems. Lately, a number of research groups10,11 has extended high-throughput studies to stem cells with these objectives: (a) understanding the conditions needed to maintain pluripotent and multipotent stem cells in an undifferentiated state while allowing self-renewal; (b) identifying and understanding the signaling pathways involved in controlling the different types of stem cells, thus gaining insights into their functioning and identifying potential targets for drug intervention; (c) understanding the differences between mouse and other species and human embryonic stem cells; (d) understanding and exploiting the potential of somatic stem cells; (e) understanding how to dedifferentiate and reprogram adult stem and other, differentiated cells. Phenotypic compound screening essentially requires the establishment of a culture system in which a cell behaves predictively and which on treatment with active compounds undergoes changes which can be readily and, preferably, automatically quantified. To test large numbers of compounds, the testing system must be amenable to automated handling. In most cases, this involves the expression of luminescent or fluorescent reporter genes and assay by either direct measurement of luminescence/fluorescent confocal microscopy or immunohistochemistry.12,13 In systems looking for therapeutic interventions, it is obvious that the screening system should mimic as closely as possible the disease state. More detailed downstream tests to validate the activity of putative “hit” molecules from phenotypic screens are also essential, for instance, gene expression profiles and functional analyses, to truly confirm production of the desired cell type. The practical considerations involved in the application of chemical biology to pluripotent embryonic stem cells and multipotent adult stem cells present different problems from those found with differentiated cells. To maintain them in an undifferentiated state, embryonic stem cells are typically cultured in media containing feeder cells, serum, and additional exogenous growth factors such as leukemia inhibitory factor (LIF) in the case of mouse embryonic stem cells and, interestingly, fibroblast growth factor-2 (FGF-2) for human embryonic stem cells. It is immediately apparent that the use of different growth factors raises questions about the use of mouse cells as models for human ones. More recently, studies have used matrices such as Matrigel and serum-free media, and Ying et al. have shown that mouse ESCs have an innate program for self-replication that does not require extrinsic instructions.14 Within culture, they can proliferate while maintaining an ability to differentiate into all the cells of the body, and these processes are well characterized as being under the control of growth factors present therein. Determining which factor(s) is responsible for the different stages of the complex differentiation process has been shown to be challenging, and there is a necessity for the use of a culture in which the stem cell is

Figure 1. Stem cell division and differentiation.

replicate indefinitely, whereas progenitor cells can divide only a limited number of times. 1.2. Stem Cell Differentiation. An embryonic stem cell can give rise to all the cell types within the body. This occurs in response to a range of signaling factors from neighboring cells and the media within the stem cell niche. Binding to these factors initiates cellular interactions which lead to activation of genes producing the proteins characteristic of the target cell type. It is implicit that this process needs to be highly selective so that only the required cell types are produced. It is possible, or more likely probable, that more than one signaling factor is involved in the differentiation process as this would increase the selectivity of the process. In any event, knowledge of the precise biochemical messengers, enzymes and/or pathways involved in the signaling process would provide targets for drug design programs. Until this is available, however, the high-throughput phenotypic screening such as cell-based and model organismbased techniques have provided the mainstay for stem cell screening. Investigation of the mechanisms of action of these compounds will provide information on the interactions involved, and this in turn will enable structure-based drug design to be initiated. This of course mirrors studies such as the case of aspirin, which was initially discovered from chemical modification of natural products without any understanding of the mechanism of action. Much later, the role of cyclooxygenase was discovered, leading to the design of specific cyclooxygenase-2 inhibitors as next-generation analgesics. C



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Figure 2. Examples of small molecules reported to manipulate stem cell fate in vitro: (a) in ESC self-renewal, (b) in reprogramming to iPSCs, (c) in dissociated ESC survival, (d) in directed differentiation of stem cells, and (e) in dedifferentiation.

reliably maintained in an undifferentiated state throughout the time period of the experiment. Only then can its expansion and/or differentiation be definitely attributed to the effect of added test molecules. An example of studies on the maintenance of stem cells is the work of Chen et al., who described a reporter assay using a

murine embryonic stem cell line from transgenic OG2 mice which had been transfected with the regulatory region of the Oct-4 gene coupled to green fluorescent protein (GFP).15 The relative levels of GFP fluorescence can be readily and automatically measured, and under incubation conditions involving the lack of feeder cells and LIF, such cells lose D



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pathways and the vulnerability of human cells to massive cell death upon single cell dissociation. As this represents a significant hurdle in the use of human ESCs, Ding screened a library of 50000 compounds to find two molecules, 9 (thiazovivin) and 10 (pyrintegrin), that significantly enhanced cell survival after dissociation while maintaining pluripotency. The authors suggest that the mechanism of action is the enhancement of cell−extracellular matrix adhesion-mediated integrin activity. In further investigations, 9 was shown to inhibit Rho-associated kinase and to stabilize E-cadherin, thus promoting E-cadherin mediated cell−cell adhesion. Thus, as well as finding reagents capable of stabilizing human ESCs following dissociation, these studies provide evidence supporting the role of cell−extracellular interactions that are presumably found within the stem cell niche in the behavior of these cells. These and other studies in the literature have shown that it is possible to maintain ESCs in culture in an undifferentiated state, thus making possible both the controlled expansion of stem cell populations and studies on the underlying biology of their controlled differentiation. 2.3. Controlled Differentiation of Stem Cells. The idea of directly influencing the differentiation of stem cells to specific cell types is particularly attractive as, in principle, the responsible compounds could be used either ex vivo to produce new cells that could be returned to the patient or directly in vivo to stimulate the regrowth to replenish damaged cells. Initial in vitro proof-of-principle studies used a mouse embryonic carcinoma cell line (P19) whose characteristics were well-known and which was relatively easy to maintain in culture. It is also multipotent and known to differentiate into cardiac muscle and neuronal cells following different specific treatments. In particular, 11 (retinoic acid) has long been known to cause differentiation to these cells toward endodermal lineages, but this agent is relatively nonspecific in target cells and its effects are hampered by chemical and metabolic instability.23 Sheng Ding et al. used this cell line and inserted into its genome a luciferase gene linked to a specific neuronal marker, Tα1 tubulin.24 This ensured that if the stem cells were to be differentiated into neuronal cells, the enhanced luciferase activity could be quantified. Hypothesizing that kinases would be involved in the differentiation process, they tested libraries based on kinase-directed molecular scaffolds and identified a number of hits including 12 (TWS119) as the most active in inducing the formation of neuronal cells. Affinity chromatography was then used to identify the enzyme glycogen synthase kinase-3β (GSK-3β) as the site of action of this agent, inhibition of which led to activation of the Wnt pathway. In a similar study, P19 embryonal carcinoma cells were transfected with the promoter region of the rat atrial natriuretic factor, a polypeptide hormone that is synthesized primarily in cardiomyocytes and is thus considered a specific cardiomyocyte marker gene, along with and upstream of the luciferase gene. Thus, an increase in luciferase activity would be indicative of differentiation to cardiac cells. A library of 100000 compounds was screened and showed a number of active compounds, of which 13 (cardiogenol C) was the most active.25 Cardiac activity was confirmed by the detection of sarcomeric myosin heavy chain, one of the essential motor neurons essential for cardiac muscle contractibility and the production of the transcription factor GATA-4, which is restricted to the

GFP expression while their compact-colony morphology completely disappeared in 4−6 days. Thus, the maintenance of GFP expression in the presence of test compounds was a good indication of the maintenance of pluripotency of the stem cells. Following screening of a 50000 compound library, compound 1 (SC1, Pluripotin) was found to be active, and further investigation of its mode of action showed it to be a dual function inhibitor of both extracellular signal-regulated kinases (ERK) and Ras GAP (Ras GTPase activating protein), pathways which, in combination, have previously been shown to play a critical role in stem cell differentiation (Figure 2).15 Thus, 1 allows the propagation of murine ES cells in an undifferentiated pluripotent state under chemically defined conditions and represented a major step forward in this field. Of course, it also showed the importance of two signaling pathways which could themselves represent targets for drug design. This work clearly addresses objective (a) and produces a reliable, reproducible system in which stem cells remain in an undifferentiated state. Initial studies on the in vitro culture of stem cells used media containing a number of high molecular weight growth factors, and a commentary by Zaret examines why the use of small molecules might be advantageous.16 This would be particularly true if the process were ever to be used on a significant scale to manufacture the amount of cells necessary for therapeutic purposes. Objectives (b−d) will be further discussed below in relation to specific organs and their diseases. Looking beyond single reporter-based readouts, much more information can be obtained in an initial screen by the use of high-content methodology in which multiple parameters associated with a desired phenotype can be simultaneously analyzed at a single cell level.17 A recent review has discussed in detail the use of advanced bioengineering techniques to produce highly integrated, accurate, and dynamically controlled cellular microenvironments in microfluidic systems in which to study stem cell self-renewal and differentiation.18 Following in vitro screening, the necessary move to in vivo studies involving zebrafish and other model organisms will be discussed in a later section. 2.2. Embryonic and Induced Pluripotent Stem Cells As Model Systems. The use of human embryonic stem cells is highly controversial, and a major breakthrough occurred with the finding that adult terminally differentiated cells could be reprogrammed to produce so-called induced pluripotent stem cells (iPSCs).19 These iPSCs were initially derived from skin fibroblasts, albeit with extremely low efficiency, and later from stomach, liver, skin, and blood. Reprogramming was carried out using the transcription factors Oct3/4, Sox2, c-Myc, and K1f4,19 but this procedure is complicated, inefficient, and carries the risk of tumor formation. Recently, a further breakthrough came with the discovery by Hou et al., who were able to replace the biomolecules by a relatively straightforward combination of seven small molecules, 2 (forskolin), 3 (2-methyl-5-hydroxytryptamine), 4 (D4476), 5 (valproic acid), 6 (CHIR99021), 7 (616452), and 8 (tranylcypromine), albeit with only 0.2% efficiency.20 It should be noted that all of these compounds have known effects on various signaling pathways. A very recent review has discussed in detail the identification of other small molecules with the ability to reprogram somatic cells to iPSCs.21 Differences in behavior between human and murine embryonic stem cells have been discussed by Xu et al.22 These differences include responses to different signaling E



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Figure 3. Roles of TPO and EPO in hematopoiesis.

• it is a clinically important system with well-established therapeutic procedures; • many aspects of the physiology, biochemistry, and cellular interactions involved have been described and more details are being determined; • the processes whereby stem cells develop into specific blood and bone cells are becoming clearer; • many constituent cell types have been characterized and in vitro assay platforms have been developed; • some potential targets for the action of small molecules have been identified and it is highly likely that more will appear as knowledge of the system increases. 3.1.1. Stem Cells Found within the Hematopoietic System. Within the bone marrow, two distinct types of stem cells are present. Haematopoietic stem cells are responsible for the maintenance of all the cells of the bloodstream, while mesenchymal stem cells are responsible for the maintenance of bone-forming osteoblasts and bone-resorbing osteoclasts. In normal health, the cellular composition of the bloodstream is maintained and renewed by the action of a small population of bone marrow resident adult hematopoietic stem cells (HSCs), with 1011−1012 mature blood cells being replenished daily. It is now clear that there exist distinct subsets of HSCs, each biased toward the production of specific groups of differentiated blood cells. In particular, it has recently been reported that, at least in mice, a platelet-biased HSC subset is responsible for producing megakaryocytes and platelets, and this also gives rise to both a myeloid-biased and a lymphoid-biased subset of HSCs. This multifunctionality would place such stem cells at the apex of the hierarchy of HSCs.29 The self-renewing HSCs occupy the top of a hierarchical population of multipotent and progenitor cells (HPCs) which differentiate along defined pathways via increasingly lineage restricted cell types in response to various signaling events, thus giving rise to all specialized functional cells of the bloodstream. True HSCs are defined in terms of functional capability, whereby they should be capable of permanently restoring and maintaining all components of the blood following transplantation into a myeloablated recipient (i.e., one suffering from severe or complete depletion of bone marrow cells). Studies in mice have indicated the presence of both long-term (LT) and short-term (ST) HSCs.30 The discovery that human HSCs can be successfully engrafted into immunodeficient mouse strains, such as the nonobese diabetic/ severe combined immunodeficiency (NOD/SCID) mouse, has allowed identification and profiling of human HSCs.31 Both mouse and human HSCs can be identified and purified using

developing heart. In addition, beating cardiac muscle cells could be visualized under the microscope. A similar strategy to the ESC study with somatic stem cells has been described by de Lichtervelde et al.26 From a library of 704 microbiological and plant natural products, 14 (euphohelioscopin A) was identified as able to selectively differentiate hematopoetic stem cells down the granulocyte/monocyte lineage. Mode of action studies suggested that 14 exhibited its activity by activation of protein kinase C. 2.4. Dedifferentiation or Reprogramming of Adult Cells. “Dedifferentiation” is a cellular process whereby a partially or terminally differentiated cell reverts to an earlier developmental stage. Li et al. have reviewed the potential role of small molecules in the reprogramming of cells such as fibroblasts to iPSCs.27 They have shown that cell fate seems to be commonly influenced by molecules that directly modulate epigenetic enzymes or mechanisms, i.e., DNA or histone modifications. During the fibroblast-to-iPSC transformation a process termed the mesenchymal-to-epithelial transition (MET) is a crucial step and involves dramatic changes in gene expression. Compounds 15 (apigenin) and 16 (luteolin) were found to upregulate E-cadherin expression and to enhance iPSC generation, again indicating the crucial role of adhesion molecules in this process.28 A point that will be obvious to medicinal chemists here is that these and indeed many other structures identified to date are likely to have numerous physiological effects and are unlikely to be very selective. Increasing selectivity will therefore be a major priority in subsequent chemical modifications. The examples above account for just a fraction of the major advances in the past several years, which are enabling efficient access to multiple cell types for in vitro screening and in which chemical biology has played a significant role. In parallel to, and in many instances building on these advances, there have been a number of seminal studies focused on discovering and developing regenerative therapies targeting specific tissues and organs.

3. STUDIES ON SPECIFIC ORGANS 3.1. Chemical Manipulation of the Hematopoietic System. The blood is one of the organs of the human body with the greatest capacity for regeneration. In addition, the use of bone marrow transplantation to treat various disorders of the bloodstream is very well established. Accordingly, one could imagine that this system offers one of the most readily translatable opportunities to the medicinal chemist. In particular: F



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Figure 4. Synthetic TPO pathway agonists and their mechanism of action. JAK 2, Janus kinase, a nonreceptor tyrosine kinase; MAP, mitogenactivated protein kinase; PI3K/Akt, phosphoinositide 3-kinase/protein kinase B; JAK/STAT, janus kinase/signal transducer and activator of transcription.

“Mesenchymal stromal cells” is an overarching term that describes multipotent cells able to produce other differentiated cells of which mesenchymal stem cells may be a subset. They are also found in the bone marrow and are responsible for the maintenance of bone-forming osteoblasts and bone resorbing osteoclasts which maintain the integrity of the adult skeleton. A further differentiation of these cells is to produce chondrocytes for maintenance of cartilage or adipocytes for lipid storage. Multipotent mesenchymal stem or progenitor cells can be used to derive in vitro populations of MSCs which exhibit the stem cell like characteristics of self-renewal and multilineage potential. MSCs are significantly less well characterized than the HSCs, with no universally accepted unique surface markers and may constitute a heterogeneous population.34 Despite their less comprehensive characterization and uncertainty as to their relevance to in vivo mesenchymal stem cells, MSCs have been used as in vitro models for the bone marrow resident cells which are considered to be responsible for the maintenance of bone, cartilage, and adipose tissues in the adult. There are also

combinations of cell surface markers, but these are not conserved between species.32 The proliferation, survival, and differentiation of hematopoietic cells is influenced by growth factors such as erythropoietin (EPO), thrombopoietin (TPO), granulocyte-colony stimulating factor (G-CSF), granulocytemacrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), and various interleukins (IL), of which EPO and TPO (Figure 3) are the most important and the most studied.33 These growth factors are expressed and secreted either constitutively by cells of certain organs (e.g., EPO by the adult liver and TPO by the liver and kidneys) or from a variety of sources in response to the body’s requirements (e.g., expression of EPO by renal cells in response to stabilization of HIF-1α (hypoxia induced transforming factor 1α) under conditions of hypoxia). Thus, interference or enhancement of the actions of these growth factors on precursor HSCs provides attractive targets for medicinal chemists interested in affecting blood cell populations. G



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receptor and stimulates multiple signaling events (Figure 4) which results in the production, through lineage commitment of the various progenitor cells, to megakaryocytes and thus to increased production of platelets. Thrombocytopenia, a serious blood-based disorder in which the number of platelets falls to a critical level, is a particularly common condition in patients undergoing chemotherapy using both traditional cytotoxic agents and with newer agents such as histone deacetylase inhibitors.40 Physiologically, it is characterized by weakness and fatigue and is often accompanied by evidence of continued bleeding from new or pre-exisiting wounds or injuries. Current treatment options for thrombocytopenia are limited and center on platelet transfusions, which are both expensive (due to an increasing move toward single patient−donor combinations) and logistically intensive (for both patient and donor), chemotherapy patients accounting for in excess of 40% of this demand. The use of recombinant TPO has been explored as a therapeutic option for thrombocytopenia, with both recombinant TPO (rhTPO, produced from CHO cells) and pegylated (PEG-rHuMGDF) derivatives being produced and advanced into clinical trials.41 Results were initially encouraging, however, the approach was thwarted by the appearance of neutralizing antibodies in 13 of the 535 healthy volunteers treated with the PEG-rHuMGDF protein, giving an alarming concomitant further drop in platelet levels. Although all of the affected subjects recovered, it is noteworthy that while no similar adverse event was noted for the rhTPO protein, the subsequent clinical development of both recombinant derivatives was discontinued. A peptide/antibody conjugate, Romiplostin (previously called AMG531, a so-called “peptibody”), has also been explored clinically as a TPO mimetic and is marketed by Amgen, having been approved in 2008.41 Upon dosing in a phase I trial, a dose dependent increase in platelet numbers was observed with no untoward immune response. However, one of the possible and dangerous side effects of the therapy is overstimulation of platelet production, leading to thrombocytosis, which may lead to blood clots and potentially fatal consequences, although to date occurrence of these type of events has been no more frequent than in placebo groups. Proteins are usually dosed intravenously, so a nonpeptidic orally bioavailable TPO mimetic was highly desirable. Toward this end, researchers at SmithKline Beecham and Ligand undertook a high-throughput phenotypic screening of their inhouse libraries.42 The assay, while being focused on an approach to a thrombocytopenia therapeutic, was not originally intended to identify compounds which bound to the TPO receptor, instead being designed to detect molecules which upregulated TPO-responsive signaling pathways, i.e., were agnostic with respect to mechanism. The readout used a luciferase reporter gene coupled to a TPO-responsive element, stably transfected into BAF-3 cells. Following the screen and deconvolution, this resulted in the identification of several classes of hit compounds, several of which and their structure− activity relationships were described in a short series of publications in 2002.42−44 The first series described, exemplified by compound 17, was at first sight unappealing to the medicinal chemist due to the presence of the diazo linker, as well as the sulfonic acid group and the naphthol unit, but structure−activity studies and subsequent transformations gave various series of compounds with improved drug-like features. The final potent nonpeptidic TPO mimetic was 18 (eltrombopag, SB497115), which

numerous reports of the clinical application of MSCs in stem cell transplantation therapies, although intriguingly in many of the instances where a benefit was noted these are believed to operate via paracrine mechanisms rather than a direct action of the MSCs themselves. Note that mesenchymal cells do not differentiate into hematopoietic cells, but interactions between the two types of cells is thought to play a significant role in influencing cell behavior in the stem cell niche, and this will be discussed later. 3.1.2. The Bone Marrow Stem Cell Niche and Signaling Pathways. The concept of a hematopoietic stem cell niche was first introduced by Schofield in 1978 as a microenvironment in which stem cell populations reside and are maintained through a variety of cues including interaction with other cell types which make up the niche.35 Subsequently, much work has been undertaken to identify the locations and components of bone marrow HSC niches, making this the best characterized niche in adult systems, although many aspects remain controversial.36 While initial investigation showed the presence of two niches, the endosteal niche found in the cellular lining between the bone and the marrow and the vascular niche found in the bone marrow sinusoids, they have come to be considered as closely connected regions of a single niche promoting and controlling the many functions of HSCs. In view of the range and importance of cells produced here, it will not be surprising to find that signaling pathways are particularly important with roles for, inter alia, VLA4 (very late antigen-4), VCAM1 (vascular cell adhesion molecule-1), CD44/hyaluronic acid, CXCR4/SDF1 (C-X-C chemokine receptor type-4/stromal cell-derived factor-1), SCF (multiprotein E3 ubiquitin ligase complex), KIT, ANG1 (angiopoetin), TIE2 (receptor for angioproteins), TPO, MPL (myeloproliferative leukemia protein), Notch,37 and Hedgehog signaling. In addition to the various chemical signals, the physical conditions within the extracellular matrix of the niche such as shear stress and rigidity have also been implicated as important for regulation and maintenance of HSCs.38 Bone marrow resident mesenchymal stem cells and their progeny, specifically adipo-osteogenic progenitor CXCL12abundant reticular (CAR) and surface cell antigen 1-expressing (SCA1+) cells, have been identified as associating closely with HSCs in the bone marrow niche and playing a key role in the regulation of HSC activity. Thus, the bone marrow HSC niche can be considered as being the same as the mesenchymal stem cell niche in which both cell types regulate the behavior of each other.39 As understanding of the important role of the niche and its components develops further, it is likely many new targets for small molecule intervention will be identified to facilitate the development of regenerative medicine. 3.1.3. Chemical Manipulation of HSCs. Because of the importance of bone marrow transplantation in a number of diseases, including leukemia and thrombocytopenia, there have been numerous studies on in vitro and ex vivo manipulation of bone marrow cells. Perhaps the most interesting study to date on agents designed to increase the levels of a blood cell type, namely the platelet, is that on small molecule analogues of TPO. TPO is a circulatory cytokine, being originally described around 50 years ago and isolated around 20 years ago. It is a 332 amino acid protein which has around 23% homology in its N-terminal region of 154 amino acids to erythropoietin. Binding of TPO to its cognate receptor c-mpl, expressed on HSCs, HPCs, and megakaryocytes, plays a pivotal role in the wound healing process. Once bound, TPO activates the H



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Figure 5. Examples of small molecules reported to manipulate cells of the hematopoietic system in vitro or in vivo.

not disclosed).48 The latter compound appears to be undergoing phase II evaluation, but neither its structure nor the trial status has been disclosed to date. What this example concisely demonstrates is that a discovery paradigm, familiar to all medicinal chemists working on “typical” contemporary drug discovery, was undertaken for a therapy which is predicated on modulation of stem/progenitor cells. A high-throughput screen was carried out using a cellbased (i.e., target-agnostic) assay system representative of the disease physiology, and the hits emerging from this were optimized following traditional structure−activity approaches. Therefore, it is clear that conventional drug discovery approaches are equally applicable to stem cell-based projects. The critical consideration is that the appropriate assay systems are available and are employed to test synthesized compounds. TPO agonism is not the only approach to the modulation of HSCs that has produced interesting results. HSCs form the basis of one of the oldest and most widely employed stem cellbased therapies, namely in the transplantation into patients suffering from bone marrow failure or myeloablative chemo/

stimulates megakaryocyte proliferation and differentiation and increases platelet counts without activating them (Figure 5).45 A histidine residue within the transmembrane domain of the TPO receptor was found to be critical for the activity of eltrombopag, leading to the hypothesis that the drug acts via binding to this domain. Moreover, this residue is only found in the TPO receptor from humans and chimps, most likely accounting for why the drug only works in these species. In so binding, it activates the JAK and STAT pathways, among other pathways, but does so in a way different from TPO or romiplostim. Eltrombopag produces much less activation of the STAT family at maximal doses than does romiplostim; unlike romiplostim, there is no activation of the Akt pathway. Eltrombopag acts at a different site on the TPO receptor, does not compete with TPO, and has been found to have an effect that is additive to that of TPO.46 Other companies have also progressed small molecule TPO mimetics to clinical trials, including Eisai (19, avatrombopag, AKR-501),47 now in phase III studies for thrombocytopenia associated with liver disease) and Ligand (LGD-4665, structure I



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Figure 6. Role of CXCR4 antagonists in promotion of HSC migration from bone marrow in vivo.

and platelet numbers with associated risk of infection or bleeding. The numbers of HSCs provided in transplant to adult recipients can be increased by use of CB from two donors, but as this increases the risk of GVHD, strategies to expand HSC populations ex vivo from CB and other sources are being sought.55 Thus, the reliable and efficient ex vivo expansion of HSC populations is a key, as yet unmet challenge which would significantly improve HSC transplantation technology, and to this end, researchers have started to develop suitable assay procedures and study pathways involved in hematopoiesis.56 For example, North et al. have described a novel highthroughput in vivo phenotypic assay using zebrafish embryos to identify small molecules which promote HSC expansion.57 A library of 2357 compounds with defined pharmacological activities was screened, leading to the identification, among other pathways, of prostaglandin E2 (PGE2) and the more chemically stable analogue 21 (16,16-dimethyl PGE2, dmPGE2, FT1050) as key enhancers of HSC expansion during zebrafish embryogenesis. Compound 21 was also found to promote recovery of kidney marrow in sublethally irradiated adult zebrafish, suggesting a role for PGE2 in adult HSC homeostasis. Further, 21 displayed similar activity in mammals, promoting expansion of mouse HSC and multipotent progenitors ex vivo and enhancing bone marrow recovery following injury with 5-fluorouracil in vivo. Expansion of human HSCs by prostaglandin E2 has also been reported,58 and 21 is currently undergoing phase I clinical trials for ex vivo expansion of umbilical cord blood derived HSCs prior to transplantation into patients with hematological malignancies (ClinicalTrials.gov ID: NCT01527838). The FDA has granted this compound orphan drug status for the ex vivo treatment of human allogenic HSCs to enhance stem cell engraftment by treating neutropenia, thrombocytopenia, lymphopenia, and anemia. This example highlights the potential value and relevance of in vivo model screens such as that of the zebrafish, which has subsequently found application in a number of other studies.59 It also highlights the potential utility of conducting screens using small molecules with defined pharmacology; they

radiotherapy for cancers such as leukemia. HSC transplantations may be autologous (HSCs sourced from recipient prior to myeloablation), which improves the prospects of successful engraftment without dangerous graft-versus-host disease (GVHD) caused by an immune response, or allogeneic (HSCs sourced from a human leukocyte antigen (HLA) matched donor) in cases such as acute myeloid leukemia (AML) where the reintroduction of the patients own cells is likely to lead to cancer relapse.49 HSCs for transplantation are harvested from three sources: bone marrow aspirates, peripheral blood, or umbilical cord blood (CB). Harvesting bone marrow is an invasive procedure usually conducted under general anesthetic and has largely been replaced by the use of peripheral blood as an HSC source.50 The number of HSCs circulating in peripheral blood is generally very low, and its use has only been made practical by the discovery that administration of G-CSF prior to harvesting promotes migration of HSCs out of the bone marrow.51 In addition to G-CSF, small molecule antagonists of the CXCR4 receptor expressed by HSCs have been developed to improve HSC mobilization (Figure 6). Compound 20 (plerixafor, also known as AMD3100 and marketed as Mozobil), a CXCR4 antagonist,52 has been shown to promote mobilization of HSCs into peripheral blood either alone or additively when administered with G-CSF.53 Compound 20 was approved by the FDA in 2008 for mobilization of HSCs to peripheral blood for autologous transplants in non-Hodgkin lymphoma and multiple myeloma patients.54 A number of next-generation CXCR4 antagonists are also reportedly in development for this indication. Umbilical cord blood has the advantage of availability from a variety of donors of different genetic background who are otherwise under-represented among donors and has been found to be more tolerant of HLA mismatches between donor and recipient than other sources. However, the small quantities of CB available from each donor has historically limited transplants to children or very small adults. Even then, the low number of HSCs leads to a delay in hematopoietic reconstitution, leaving recipients with reduced immune cell J



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that this compound acts by inhibition of the platelet-derived growth factor receptor (PDGFR), leading to promotion of megakaryocyte/erythrocyte progenitor (MEP). A modest increase in megakaryocytes at the expense of erythrocytes was also observed when MEPs were cultured in the presence of 24 but in the absence of EPO. 3.1.4. Chemical Manipulation of MSCs. The ex vivo expansion of MSCs has not been examined to the same extent as has been the case with HSCs, and there are no small molecules which have so far been found to affect this expansion. Ex vivo MSC differentiation, however, has attracted more interest and, for example, Schultz et al. used cell-based highthroughput screening against mouse embryonic mesoderm fibroblast C3H10T1/2 cells to identify small molecules capable of promoting osteogenesis.66 These cells are multipotent mesenchymal progenitors which have been used as readily available, homogeneous, and easily cultured alternatives to bone marrow derived MSCs (BM-MSC) because they are able to differentiate into osteoblasts, chondrocytes, and adipocytes, although their adipogenic potential has been shown to be lower than that of MSCs.67 From a 50000 compound library, 25 (purmorphamine) was found to be capable of promoting osteogenesis of murine C3H10T1/2 cells. Subsequently, it was shown to promote osteogenic differentiation of human BMMSCs68 to enhance the osteogenic activity of BM-HSC derived osteoblasts ex vivo69 and to act synergistically with bone morphogenetic protein 4 (BMP-4).70 Further studies indicated a mechanism of action of 25 involving activation of the Hedgehog (Hh) signaling pathway through agonism of the transmembrane protein Smoothened (Smo),70 highlighting a critical role for the Hedgehog signaling pathway in osteogenesis. Screening protocols using human BM-MSCs have also been described. Burdick et al. screened 1040 small molecules against human MSCs cultured in osteogenic medium and identified a number of compounds which either inhibited or promoted osteoblast differentiation,71 while Alves et al. reported a highthroughput phenotypic screen to identify compounds capable of enhancing osteogenic differentiation of human BM-MSCs when cultured in the presence of dexamethasone.72 A number of hit compounds were identified, with alkaline phosphatase (ALP) activity normalized to cell number again being used as a measure of osteogenesis. Interestingly, the efficacy of hit compounds was found to vary when MSCs derived from different donors were used. This observation highlights that while screens against human BM-MSCs may be more relevant to understanding or developing treatments for human diseases compared to screens against mouse C3H10T1/2 cells, the results may be less reproducible due to heterogeneous MSC populations and donor variation. Of the hit compounds, 26 (H8) was identified as capable of upregulating ALP activity in MSCs derived from the four donors tested, either alone or synergistically with dexamethasone. Compound 26 has also been identified as an ATP competitive inhibitor of protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), and myosin light chain kinase (MLCK).73 However, the authors show that, consistent with previous reports,74 cyclic adenosine monophosphate (cAMP), the endogenous activator of PKA, promotes rather than suppresses ALP expression, leading to the suggestion that 26 is acting through an as yet unidentified PKA-independent mechanism to promote osteogenesis.

may yield useful insights into mechanisms and possible targets for future drug discovery. However, many pathways and components are either not conserved or show differences between zebrafish and other systems, which can make translation of findings to humans challenging and a potential limitation of this screening strategy. Boitano et al. identified 22 (StemRegenin 1, SR1) from screening 100000 heterocyclic molecules in a cell-based assay assessing ex vivo expansion of primary human CD34+ cells derived from HSC mobilized peripheral blood.60,61 Ex vivo culture in the presence of 22 also significantly expanded populations of CD34+ cells derived from human umbilical cord blood compared to control, and these expanded populations were capable of long-term engraftment into NOD/SCID mice with greater efficiency than nontreated cells, or cells expanded without 22. Compound 22 was identified by its effect on the expression of two genes known to be under the control of the aryl hydrocarbon receptor (AhR). Compound 22 was found to act as an (AhR) antagonist, thus identifying a new molecular target for small molecule agents to promote HSC expansion ex vivo. Interestingly, 22 did not promote expansion of mouse HSCs ex vivo due to lack of activity at the murine AhR. This highlights the importance of considering the underlying crucial differences between human and murine cells. Other pathways have also been implicated in HSC expansion and this knowledge exploited to identify small molecule modulators. For example, inhibition of p53 activity has been employed by Leonova et al. as a targeted strategy to expand mouse HSCs.62 p53 has previously been shown to regulate selfrenewal of HSCs/HPCs,63 and it was found that administration of the p53 inhibitor 23 (pifithrin β) to mice increased numbers of HSCs in vivo and also promoted their mobilization into the peripheral blood. In addition, 23 was also found to expand populations of mouse HSCs/progenitors when added to in vitro cultures. Of course, in these types of approaches, it has to be established that the molecule is selective and that the observed effect is indeed solely due to the identified mechanism. Inhibition of p53 may seem to be a questionable strategy for expansion of stem cell populations in vitro or in vivo with the intention of therapeutic application given its critical tumor suppressor role. However, in this study, Leonova et al. show that administration of a single dose of 23 to p53± heterozygous tumor susceptible mice prior to a dose of total body ionizing radiation did not lead to increased tumor incidence compared to control. This result, in combination with a cited study by Christophorou et al.,64 led the authors to suggest that short-term inhibition of p53 does not lead to increased tumor incidence and that p53 inhibition represents a valuable strategy for HSC expansion. As with other studies relating to modulation of stem cell function, appropriate early stage safety studies would be critical to test this hypothesis. Small molecules have also been identified which promote differentiation of HSCs or multipotent progenitors to particular hematopoietic cell types although these have yet to be applied in vivo. Compound 24 (MK1) was identified following a highthroughput screen of a 50000 member library for compounds enhancing differentiation of primary human mobilized peripheral blood derived CD34+ HSCs to CD41+ megakaryocytes.65 Such control over differentiation could be used to generate populations of megakaryocyte progenitors for inclusion in HSC transplants to alleviate platelet deficiency and risk of hemorrhage prior to full hematopoietic reconstitution by the transplanted HSCs. Further biochemical investigations showed K



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The translation of basic stem cell science to a clinically used agent in the discovery of eltrombopag and plerixafor represents highly significant milestones and achievements and gives rise to optimism that similar achievements can be made in other organs. 3.2. Chemical Manipulation of Neuronal Regeneration. Damage to the central and peripheral nervous system has been estimated to affect perhaps 1 billion people worldwide and is thus a major cause of human suffering. In view of the aging population, it is predicted that these numbers can only increase in the future. These disorders include Parkinson and Alzheimer’s diseases and less common conditions such as motor neuron disease, multiple sclerosis, and rare diseases such as spinal muscular atrophy. Also, there are numerous psychiatric conditions which may involve neuronal damage. Clearly, there is a pressing need for novel therapies able to alleviate the resultant suffering. Neural stem cells (NSCs) and neural progenitor cells (NPCs) were first detected in 1989, and their existence and potential are now widely accepted.81 NSCs are multipotent cells that generate neurons, astrocytes, and oligodendrocytes,4 and while they were initially identified in the subventricular zone (SVZ) of the mouse brain, neural progenitor cells capable of the production of specific neuronal lineages have been described in other regions including the dentate gyrus of the hippocampus and hypothalamus. NPCs differ in that they are oligopotent and do not self-renew indefinitely but appear to exist between NSCs and differentiated cells. The balance between the two cell types appears to be maintained by an interaction between the epidermal growth factor (EGF) receptor signaling pathway and the Notch signaling pathway.82 Neurogenesis is the process whereby neurons, and the glial supporting cells, are generated from NSCs and NPCs and thus provides a route by which lost or damaged neural cells can be replaced. The stimulation of this process would thus be therapeutically valuable. It is hoped therefore that NSCs and NPCs could be stimulated to replace damaged neurons with functioning, normal ones and that this approach may also provide a route to the treatment of conditions resulting from trauma injury to the CNS. The studies described below will show that this hope is not without foundation. Several potential methodologies are being explored including the manipulation of NSCs and NPCs in vitro to generate tissue types for disease modeling and drug efficacy and toxicity testing, stem and progenitor cell therapy approaches involving ex vivo stimulation, and differentiation followed by transplantation into the patient and in situ stem cell modulation. A particular technical hurdle exists, however, in that obtaining neuronal stem cells from the patient is not easy and it is likely that in situ modulation would be more clinically acceptable. Alternatively, dedifferentiation or trans-differentiation of more readily available adult cells into neuronal cells would seem to be an attractive approach. In any event, the use of small molecules to produce the desired cell type from a number of precursors has made great progress. 3.2.1. In Vitro Production of Neural Cells. Historically, neuronal stem cells have proved difficult to culture in vitro as single cells and most work has involved the use of neurospheres, i.e., 3D aggregations of free-floating clusters of cells which contain only a small percent of neural stem cells along with progenitor cells and growth factors such as EGF and FGF. Withdrawal of growth factors activates differentiation into

cAMP/PKA activation has previously been shown to promote osteogenic differentiation of human BM-MSCs in studies using the cell permeable cAMP analogue 27 (N6,2′-Odibutytyadenosine,3′,5′ cyclic monophosphate, db-cAMP)75 although conflicting results were obtained with rodent MSCs and osteoblast progenitors in which treatment with cAMP, 27, or another analogue, 8-bromo cAMP (8b-cAMP), led to inhibition of osteogenic differentiation and bone formation. Laurencin et al. evaluated the effect of the more recently developed cAMP analogue N6-benzoyladenosine-3′5′-cyclic monophosphate (6-Bnz-cAMP), a PKA-selective cAMP analogue, on mouse osteoblast-like MC3T3-E1 precursor cells.76 It was found that, in contrast to treatment with cAMP or nonselective analogues, in vitro treatment of MC3T3-E1 cells with 6-Bnz-cAMP led to increased expression of the osteoblast specific transcription factor Runx2, enhanced ALP activity, promotion of osteopontin and osteocalcin production, and matrix mineralization. Treatment with 6-Bnz-cAMP led to an increase in phosphorylation of the PKA substrate cAMP response element-binding protein (CREB), consistent with activation of PKA and suggests a role for PKA activation in osteogenesis.77 Further support for the role of PKA activation in osteogenesis is provided by the work of Suh et al.78 Compound 28 (CW008) was derived from chemical optimization of a hit compound identified in a high-throughput cell-based screen designed to identify promoters of osteogenesis in human BMMSCs. As well as promoting osteoblast differentiation, 28 was also reported to suppress adipogenesis. Compound 28 was found to act through induction of cAMP production, so activating cAMP/PKA/CREB signaling, leading to increased levels of phosphorylated CREB (pCREB). It was also found that 28 significantly reduced expression and secretion of the adipocyte derived hormone leptin, which may play a role in the regulation of osteogenesis. In addition, 28 displayed osteogenic properties in vivo, leading to significantly increased bone mass and bone volume density when administered to ovariectomized (OVX) mice, which serve as a model for estrogen deficient osteoporosis. Gwak et al. identified small molecules driving osteoblast differentiation, reportedly through upregulating expression of the key transcription factor Runx2.79 From a 270000 compound library tested against a stably transfected HEK293 reporter cell line, 29 (SKL2001) was identified as a specific activator of Wnt/β-catenin signaling.80 Intracellular β-catenin levels are controlled by binding to the scaffolding protein Axin, which also interacts with the kinases GSK-3β and CK1, thereby promoting the selective phosphorylation of β-catenin at sites which mark the protein for ubiquitination and proteosomal destruction. Compound 29 was found to inhibit the interaction between β-catenin and Axin, thus preventing β-catenin phosphorylation and promoting cellular accumulation. When mouse multipotent mesenchymal ST-2 cells or human BMMSCs were exposed to 29, osteoblast differentiation was promoted and differentiation of 3T3-L1 preadipocyte cells was suppressed, consistent with upregulation of Runx2 activity by Wnt/β-catenin agonism. The hematopoietic system is perhaps the best studied and understood one, and clinically useful transplantation procedures using bone marrow extracts is well established. Thus, it is perhaps not surprising that this is the area in which greatest progress has been made on identifying small molecules with the capacity to promote in vivo cell expansion and differentiation. L



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Figure 7. Examples of small molecules reported to manipulate neuronal cells in vitro and in vivo.

the final cells. The heterogeneous cell populations within these structures makes them less than ideal, and recently, Theus et al. has published a detailed procedure for the preparation of cultures of adherent monolayer cells which may act more consistently.83 The signaling pathways involved in NSC selfrenewal and differentiation are becoming clearer, with EGF, Hedgehog, vascular endothelial growth factor, BMP, Notch, and LIF-JAK-STAT identified to date. 3.2.1.1. Directed Differentiation of NSCs and NPCs in Vitro. The task of inducing neuronal differentiation in a culture of NSCs is essentially to prevent self-renewal and to initiate differentiation, i.e., modulation of the appropriate signaling pathways and a number of high-throughput screening studies

have produced small molecules which can do just that. For example, Lange et al. showed that inhibitors of glycogen synthase kinase-3 (GSK-3) enhance neuronal differentiation in human NPCs.84 The two most effective compounds were 30 (SB21, SB216763) and 31 (kenpaullone), which, among other effects, both activate the canonical Wnt signaling pathway (Figure 7). Warashina et al. also showed that 32 (neuropathiazol) was able to enhance neurogenesis while suppressing astrogliogenesis in rat NPCs, albeit via an unknown mechanism.85 3.2.1.2. Directed Differentiation of ESCs and iPSCs. It has been found that ESCs grown on monolayers in the presence of LIF (leukemia inhibiting factor), and the small molecules 6, an M



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Table 1. Selected Antidepressants and Corticosteroids and Their Role in Neurogenesis in Vivo

inhibitor of GSK-3, and 33 (SB431542), a TGF-β inhibitor, produces primitive NSCs which can stably self-renew and retain potential and responsiveness to instructive neural patterning cues toward midbrain and hindbrain neuronal subtype. Remarkably, these pNSCs could be transplanted into the lateral ventricle of neonatal mice and were subsequently found to be distributed in many brain areas, including the corpus callosum, the subcallosal zone, and the caudate-putamen.86 Compound 34 (icaritin), which has been reported to possess antiaging activity, was found to increase the differentiation of mESCs into cholinergic neurons in vitro.87 The same group subsequently found that 35 (isobavachin) induced the differentiation mESCs into neuronal cells reportedly by a protein prenylation mechanism.88 Similarly, human ESCs grown in the presence of noggin, a protein that inhibits signal transduction of the TGF superfamily produce neuroectoderm, which in the presence of β-FGF/EGF give neurospheres. Treatment of these with 36 (Y27632) produces increased levels of migrating NC-like cells. These were able to colonize explants of embryonic mouse gut and from there to differentiate into enteric neurons. This could represent an approach to the treatment of some neurocristopathies.89

The urinary analgesic drug 37 (phenazopyridine) has been found in a high-throughput screen to increase total neurite outgrowth from human ESCs and to produce a homogeneous neuronal precursor cell population. 90 After one week, glutamatergic, GABAergic, and tyrosine hydroxyase-positive neurons were seen, while after four weeks, additionally nestin, β3-tubulin, SOX1, and vimentin-positive neurons were detected along with mature neurons and astrocyte and oligodendrocyte progenitors. A complementary finding to the above result is that of Han et al., who showed that the antiarrhythmic drug 38 (amiodarone) is selectively toxic to NSCs but not dopaminergic neurons derived from human ESCs. In principle, this would provide a way of producing a purer enriched population of neurons,91 an interesting example of unexpected findings from existing drugs apparently acting in different tissues. The generation of neurons from stem cells will only be of use if the resultant neurons have the properties of native cells. A recent review has detailed the evidence that spinal motor neurons and cortical pyramidal neurons derived from stem cells show a remarkable degree of fidelity with normal embryonic development.92 Similarly, another detailed investigation of spinal motor neurons generated from human stem cells shows N



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3.2.2. In Situ Stem Cell Modulation. 3.2.2.1. Small Molecules Promoting Neurogenesis in Vivo. It is an intriguing thought that existing drugs might exert some or all of their effects by promoting neurogenesis or by some other effects on brain cells. The relationships between neurogenesis and the effect of antidepressants has been reviewed (Figure 5).103 Importantly, antidepressants from multiple mechanistic classes have all been shown to induce neurogenesis in vivo, including selective serotonin reuptake inhibitors (SSRIs) such as 43 (sertraline) and 44 (fluoxetine), noradrenalin reuptake inhibitors (NRIs) such as 45 (imipramine) and 46 (reboxetine), and monoamine oxidase inhibitors (MAOIs) such as 8 (Table 1). Duman reviewed the state of knowledge in 2004 and showed that levels of brain-derived neurotrophic factor in brain regions associated with depression are reduced following stress. Furthermore, antidepressant treatment reverses this and the article reviews the work leading to a neurotrophic hypothesis of depression and antidepressant action.104 However, the association between neurogenesis and antidepressant action remains controversial. It is important to note that studies have shown that the effect of such agents is restricted to one NSC nest within the brain, they are not pleiotropic effects on the resident stem cell populations, which fuels the hope that drugs with selectivity can be developed in future. Antidepressants such as 43 have been shown to increase adult human hippocampal neurogenesis at least in part via a glucocorticoid receptor dependent mechanism which requires PKA signaling, glucocorticoid receptor phosphorylation, and the activation of a specific set of genes.105 Likewise, chronic treatment of middle-aged rats with 44 increases the volume of the hippocampus and increases the expression of molecules related to structural plasticity and inhibitory neurotransmission. Similar but lesser effects are seen in the amygdala.106 Intriguingly, when tested in vitro, sertraline alone did not induce neuronal proliferation, but when coadministered with 47 (cortisol), an effect that was reversed by cotreatment with 11β-[p-dimethylaminophenyl]-17β-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one (RU-486, Mifipristone), consistent with a glucocorticoid receptor-dependent mechanism.105 This observation leads to an intriguing paradox: while this effect could help to account for the observed selectivity for one NSC nest within the brain, it raises questions of how in vitro screening systems can best be designed to detect compounds acting in a similar manner. Clearly, as our understanding of the components and interactions within the stem cell niche evolves, in vitro models can be constructed to better reflect the in vivo situation. Acute traumatic brain injury (TBI) is a leading cause of disability and death, and currently there is no approved clinical treatment. Histone deacetylase inhibitors (HDACis) have been shown to be effective neuroprotective agents following TBI in rodent models, however, there are at least four classes of these enzymes and it is not clear if the protective activity is due to a particular subtype. One particular compound, 48 (LB205), has been shown to operate by preservation of the nerve growth factor (NGF)/tyrosine kinase receptor type 1 (TrkA) pathway and augmentation of proliferation of nestin-expressing cells with stem cell characteristics.107 Compound 49 (allopregnanolone), a metabolite of progesterone, is neuroactive and is a potent in vitro inducer of neural progenitor proliferation of both human and rodent derived cells. In vivo, 49 significantly increases neurogenesis in the

expected morphological and electrophysiological developments along with neurite outgrowth and increased soma area.93 Experiments of this nature will form a critical part of the downstream assessment of compounds modulating stem cell function. Sixteen iPSC lines isolated from seven individuals were shown to give functional motor neurons with a range of efficiencies similar to that of human ESCs, showing the potential utility of these cells.94 Recently, the group of Wichterle have published a detailed account of their method of converting human stem cells, both embryonic and induced pluripotent, rapidly into motor neurons with defined subtype identities of relevance to the study of neurodegenerative disease.95 A different combination of inhibitors, namely 39 (DMH1) and 33, has been claimed to give specific and independent inhibition of BMP and TGF-β1 pathways in human iPSCs.96 Compound 39 in conjunction with 40 (SB218078) has recently been shown to induce human iPSCs grown on Matrigel in N2B27 medium supplemented with primitive neuroepithelium to produce a highly enriched population of neural stem cells with concomitant down-regulation of markers for pluripotency. These cells were reported to be particularly suitable for the formation of dopaminergic cells.97 3.2.1.3. Directed Differentiation of Bone Marrow− Mesenchymal Multipotent Stromal Cells. A small molecule inhibitor of the PI3K(phosphatidylinositol 3-kinase)/AKT signaling pathway that can inhibit and promote neuronal differentiation of rat mesenchymal multipotent stromal cells has been described. Compound 41 (LY294002), an inhibitor of this pathway, inhibits MSC proliferation, directs the neuronal differentiation of MSCs, stimulates FAK (focal adhesion kinase) phosphorylation, and increases cell adhesion.98 Several compounds which inhibit this target have been progressed into the clinic, and it would be very interesting to assess their activities in these systems. 3.2.1.4. Differentiation of Other Cell Types. An issue which has arisen in trials in model systems transplanting ESC- and iPSC-derived neuronal cells is the potential for uncontrolled growth and tumor production in vivo ascribed in part to the proliferative capacity of precursor cell types. A means to circumvent this issue would be to derive cells directly from nonproliferative/less proliferative cells, e.g., directly from fibroblasts. To this end, Vierbuchen et al. used Ascl1, Brn2, and Myt1l on mouse fibroblasts to produce neurons,99 while Son et al. have described how fibroblasts from both the mouse and the human can be converted to induced motor neurons (iMNs) using a limited number of selected transcription factors, namely seven (Ascl1, Brn2, Myt1l, Lhx3, Hb9, Isl1, and Ngn2) in the mouse and one more, NEUROD1, in the human.100 Interestingly, this conversion process does not appear to involve a neural progenitor state but the resultant neurons behave as normal neurons and are susceptible to damage from glial cells derived from diseased tissue. A member of the forkhead class O (FoxO) proteins, FoxO3a has been shown to be a critical regulator in the reprogramming of mouse embryonic fibroblasts to iPSCs and thence to NSCs.101 A few years ago, a small molecule 42 (neurodazine) was reported to induce transdifferentiation to a neuronal cell type from myoblasts, albeit in vitro.102 Together, these findings open up an intriguing possibility that it may be possible to develop small molecules to induce transdifferentiation to the desired cell type from a nonstem or progenitor cell type in vivo. O



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neurogenesis of NPCs at the expense of proliferation in the subgranular layer of the dentate gyrus. Schneider et al. have reported on the mechanism of action of a series of isoxazoles, compounds originally identified as in vivo active agents which promoted cardiomyogenesis in response to injury (see section 3.4.2.2) and shown a crucial role for a protein GPR68, a proton sensing G protein-coupled receptor with no previously known function in the brain.117 Compound 57 (Isx) and GPR68 coregulated neuronal target genes such as Bex1 (brain-enriched X-linked protein-1) in hippocampal progenitor cells. In vivo, 57 promotes neurogenesis in type 1 NSCs and after traumatic brain injury cerebral cortical astrocytes express GPR68, indicating a crucial role for the effect of brain pH. The authors speculate that lactic acidosis could explain the beneficial effect on Alzheimer’s disease seen following exercise. Microarray gene expression analysis showed that 57 promoted upregulation of genes associated with neurogenesis and cell cycle progression in primary mouse dentate gyrus cells in vitro, consistent with conversion of quiescent type 1-NSCs converting to proliferative transit amplifying progenitors prior to terminally differentiated neurons. Compound 57 was found to be a blood−brain barrier penetrant and able to promote enhanced hippocampal neurogenesis when administered by intraperitoneal injection to young mice, with an associated decrease in type 1-NSC populations. Despite increased neurogenesis, as assessed by dentate gyrus volume combined with nestin-GFP+ cell count and cell lineage tracing, 57-treated mice did not display any learning or memory enhancement over controls in established behavioral tests. This work suggests a link between pH, which is often linked to hypoxia, and regulation of neurogenesis. This link may also form part of the co-ordination of the body’s response to injury and provides an opportunity for small molecule therapeutic intervention. 3.2.2.2. Manipulation of Neuronal Cell Migration in Vivo. A recent, detailed review has discussed neurogenesis in both embryonic and adult zebrafish.118 It appears that in the embryo neurons are derived from the neuroectodermal epithelium while in the adult they originate from the glial cells and need to be incorporated into existing functional tissue. Clearly, a better understanding of these mechanisms could lead to the discovery of agents able to influence the migration of neurons into functioning brain structures. Methods of imaging the neural migration, axonal, and dendrite formation in this species have also been described.119 Zhang et al. have shown that Ncadherin maintains β-catenin signaling via AKT (protein kinase B) activation in mouse cortical development.120 Inhibition of AKT signaling in neural precursors in vivo leads to reduced βcatenin-dependent transcriptional activation, increased migration from the ventricular zone, premature neuronal differentiation, and increased apoptotic death. The growth factor, IGF-1 (insulin-like growth factor-1), has been found to act on SVZ-derived adult neuronal precursor cells to induce migration along the rostal migratory stream (RMS) through both chemokinesis and chemotaxis.121 This suggests that the aforementioned pathways may represent new targets for increasing precursor cell recruitment into areas of cellular damage resulting from brain injury or illness. Jiang et al. showed that the synthetic cannabinoid HU-210 and the endocannabinoid anandamide are able to promote hippocampal neurogenesis. 122 Both appear to act via cannabinoid receptor 1 (CB 1 R) and to promote the proliferation of embryonic hippocampal neural stem/progeni-

subgranular zone of the dentate gyrus and the subventricular zone of the 3xTgAD mouse model. A once a week dosing schedule seems the most efficacious, and this molecule is currently the only small molecule with the ability to promote neurogenesis and to reduce AD pathology burden.108 The discovery of small molecules to promote the expansion, survival, differentiation, and/or migration of neural stem/ progenitor cells using small molecule agents represents an attractive strategy to tackle neurodegenerative disease or promote repair following CNS injury. Two such studies are those of Teo et al., who showed that 50 (ICG-001), a selective inhibitor of β-catenin/cAMP-response element binding protein (CREB)-binding protein, corrects defects in neuronal differentiation in a preclinical model of Alzheimer’s disease,109 and Schultz et al., who reported that M1/M3 muscarinic receptor antagonist 51 (benztropine) shows significant decrease in disease severity in a clinically relevant mouse model of multiple sclerosis through increasing remyelination.110 However, while a number of small molecule agents capable of promoting neurogenesis in vitro have been identified, extension to the in vivo situation involves additional challenges associated with optimization of pharmacokinetic properties and specifically in the neurological case, penetration of the blood−brain barrier. Compound 52 (P7C3) and a number of analogues including 53 (P7C3A20) have been discovered in an in vivo test following the development of hippocampus neurogenesis in newly born mice. A large proportion of neurons die during the first week of life in the subgranular zone of dentate gyrus, and these molecules are able to diminish this.111 Even more interestingly, the same compounds are able to protect adult mice against the effect of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a known neurotoxin, especially against dopaminergic neurons.112 This robust protection of mature dopaminergic neurons is also seen in the worm. Both compounds, but especially 53, also protects ventral horn spinal cord motor neurons from cell death in the G93A-SOD1 mutant model of ALS.113 As these results are found in vivo in mature mice in an accepted model of Parkinson’s disease, they are potentially extremely exciting and the authors state that they are currently examining other compounds which are likely to have improved pharmacokinetic profiles and reduced potential for toxicity. The effect of hypoxia on neural development has been addressed by a number of authors and in a detailed review.114 A crucial role for cGMP in the in vivo differentiation of NSCs to neurons in rat prefrontal cortex and hippocampus was shown by Gómez-Pinedo.115 Treatment of pregnant rats with 54 (LNAME), which significantly reduces levels of cGMP in the fetus brain, results in reduced proliferation to neurons and increased proliferation to non-neuronal cells. This effect was reversed by the concomitant treatment with 55 (sildenafil). Wurdak et al. developed compound 56 (KHS101) by chemical optimization of thiazole-based hits identified in a phenotypic screen for compounds able to promote neuronal differentiation of rat hippocampal NPCs. 116 Chemical proteomics studies identified transforming acidic coiled-coilcontaining protein 3 (TACC3) as a protein target bound by KHS101, which was proposed to lead to increased nuclear localization of the transcription factor ARNT2. As well as promoting differentiation of NPCs to neurons, 56 also suppressed NPC proliferation and BMP4 induced astrogenesis in vitro. When administered to rats by subcutaneous injection, 56 was found to cross the blood−brain barrier and to promote P



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Figure 8. Effect of cannabinoid receptor modulators on neural stem/progenitor cell proliferation and neuroblast migration.

3.2.3. In Vitro Disease Models, Screening, and Future Drug Discovery. Medicinal chemistry approaches to neuropathies could be greatly changed by the ability to produce neurons and glial cells which could be used in high-throughput screening. A major advance was provided by the groups of Eggan and Wichterle when they showed that human iPSCs generated from patients with amyotrophic lateral sclerosis (ALS) could be differentiated into motor neurons, thereby giving rise to expectations that the molecular details of the disease could be studied and potential treatments identified.125 Gaspard and Vanderhaeghen have discussed how disease specific human iPSCs generated from patients might be used to screen for small molecules able to repair the damaged neurons.126 The problems of such approaches to “in vitro disease modelling” have been discussed in thoughtful and comprehensive reviews by Sandoe, Merkle, and Eggan.127 In particular, these show how detailed studies of the cell-type-specific gene expression of the damaged neurons could lead to an understanding of the true molecular nature of the disease and consequently to the discovery of agents able to correct or alleviate this. Further, they discuss potential problems in the technology due to variability in human iPSCs isolated from individual patients, the

tor cells without significant effects on differentiation, thus resulting in more newborn neurons. Subsequently, Doherty showed through the use of arachidonyl-2′chloroethylamide and JWH-133 that endocannabinoid signaling in mouse brain is required for neuroblast migration from the subventricular zone.123 This effect was also seen on treatment with cannabinoid receptor agonists and has given rise to the hypothesis that anxiolytic and antidepressant-like effects of cannabinoids arise via promotion of hippocampal neurogenesis (Figure 8). 3.2.2.3. Reprogramming in Vivo. Lie et al. have shown that in the adult mouse hippocampus expression of the SoxC transcription factors Sox4 and Sox11 is initiated at around the time of neuronal commitment to adult NSCs and is maintained in immature neurons. In addition, both of these factors in combination with Ngn2 reprogram astrocytes into neurons in vivo.124 Future deconvolution of these complex pathways controlling NSC differentiation and neural cell identity should lead to the discovery of new targets which may potentially enable the chemically mediated reprogramming of neuronal somatic cells. Q



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number of unique cell types estimated as being in excess of 50. From a developmental perspective, cell fate determination and differentiation is influenced by both local cell−cell contacts along with paracrine signaling mechanisms. Multipotent retinal progenitor cells ultimately give rise to the various cell types that comprise the retina136 mediated by a variety of signaling pathways. Of particular note, signaling through Notch/Delta and Hedgehog having been implicated, with growth factors such as FGF also having been shown to play an important role in differentiation to photoreceptor cells.137 Interestingly, transdifferentiation of the retinal pigment epithelium (RPE; a single layer of hexagonal closely packed pigmented cells) has also been demonstrated in a number of studies in model systems, illustrating that lineage plasticity, as with other tissue types and cell populations, is still maintained even in a mature cell lineage. Importantly, from a drug discovery perspective, various assay systems have also been described which have the potential to serve as model systems for assessing the impact of agents on the various stages of retinogenesis from human ESCs and iPSCs.138 Degeneration of the retina is one of the most significant causes of blindness in both the developed and developing world and, accordingly, is a major concern from both healthcare and economic perspectives. Contrary to general perception, retinal degeneration is a disorder that affects not only the elderly but rather is common across all age groups. Of the disorders of the retina, among the most prevalent and well-known are retinitis pigmentosa (RP), diabetic retinopathy (DR), and age-related macular degeneration (AMD). RP affects primarily younger adults and children and is caused by abnormalities in and loss of the light sensing photoreceptor cells. DR is a disorder most prevalent in middle aged adults in which retinal blood vessels become leaky and gradually degrade and rupture. Finally, perhaps the best known retinal disease is AMD, in which cellular debris accumulates between the choroid (the central layer at the back of the eye which contains the blood supply) and the retina. This ultimately results in retinal detachment and is commonly found in the older section of the population. AMD is a particularly widespread and devastating visual disease, with approximately 14 million people suffering from it worldwide.139 Currently there are three treatment options for patients suffering from these type or disorders: pharmacological or surgical treatments and cell replacement therapies. Pharmacological approaches are most commonly used for AMD and include the anti-VEGF biological agents Lucentis, Macugen, and Avastin, all of which are delivered by intraocular injection. Anti-VEGF therapies have also been used for diabetic retinopathy. Other than the use of corticosteroids in diabetic retinopathy,140 there are currently no small molecule agents approved for these indications which tackle the underlying disease pathology, although there are a number of VEGF-R inhibitors in clinical development for other indications, with the potential for off-label use in retinal disease. Surgical approaches, including transplantation of RPE cells (to treat AMD), has been used with limited success to date, and laser surgery is an established treatment paradigm used for diabetic retinopathy. Accordingly, the opportunity to unleash the power of stem cell based therapeutics has clear potential for the treatment of these diseases.141 One of the first therapeutic areas to benefit from the discovery and evolution of stem cell therapeutics has been retinal disease, where a number of companies and organisations

variable behavior of such cells in culture, and epigenetic variations. They do conclude, however, that recent advances in reprogramming techniques is allowing the production of large cohorts of disease and control pluripotent cell lines which should reduce the possibility of results being distorted by the presence of aberrant cell lines. A similar discussion has been presented by Yung et al.128 Studies recorded to date include Neuropathic Gaucher’s disease using patient derived iPSCs,129 neurodegeneration with stem cell derived motor neurons and astrocytes with activated microglia,130 Parkinson’s using iPSC-derived dopaminergic neurons131 and neuroactivity/neurotoxicity with stem cells under the control of neural cell specific tetralin-α promoter.132 Chung et al. have shown that cortical neurons can be generated from iPS cells of patients harboring mutations of a key protein involved in Parkinson’s disease, α-synuclein, and identified a small molecule, 62 (NAB2), which affects the ubiquitin ligase, Nedd4, and is able to reverse the pathologic phenotypes in these neurons.133 ALS is a rapidly progressing neurodegenerative disease which is characterized by the death of motor neurons. Today, there are unfortunately no effective treatments so the discovery of agents capable of preventing motor neuron death would represent a major step forward. Recently, Yang et al. have described a high-throughput screen using motor neurons from wild-type and mutant SCOD1 mouse ESCs which has shown that the heterocyclic alkaloid 31, a dual inhibitor of GSK-3 and HGK (which is also known as MAP4K4) kinase is able to prolong the healthy survival of both types of motor neurons and, most interestingly, improved the survival of human motor neurons derived from ALS-patient-induced PSCs.134 As reproducible cell lines are essential for screening of candidate compounds, a technique for the differentiation of human ESCs and iPSCs using small molecules including 33 (dorsomorphin), 6, and 25 to early neural progenitors (small molecule neural precursor cells, smNPCs) has been described. smNPCs are robust, exhibit immortal expansion, and have the potential to clonally and efficiently differentiate into neural tube lineages, including motor neurons and midbrain dopaminergic neurons as well as neural crest lineages, including peripheral neurons and mesenchymal cells.135 Perhaps the most interesting and thought-provoking results from the studies on in vivo small molecule modulation of neurogenesis is the fact some well used, and perhaps wellcharacterized, drugs may have effects on neurogenesis. Whether or not this is relevant to their clinical activities will only become clear with future work, but it does underline the belief that small molecules may be discovered to safely influence cell proliferation, differentiation, migration, and functional integration in vivo in patients. Of course, for neuronal applications, the development of any active molecules comes with the additional challenge of manipulating biodistribution, particularly with respect to CNS penetration. Nonetheless, a number of seminal studies have been published in recent years establishing in vivo proof-of-principle in animal models and it remains to be seen whether these new discoveries will translate to the clinic in due course. 3.3. Chemical Manipulation to Effect Retinal Regeneration. 3.3.1. The Retina. The retina is the light sensitive layer of cells within the inner surface of the eye which is responsible for vision. The general structure and overall organization of the retina is well conserved across vertebrate species and consists of seven classes of cells, with the total R



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Figure 9. Examples of small molecules reported to manipulate retinal cells.

due to modulation of the Wnt and Nodal pathways, respectively. The resulting hES-derived retinal progenitor cells were shown as being competent to differentiate into RPE and photoreceptor cells, with this differentiation being induced by treatment of the cell culture with retinoic acid and taurine. Finally, the same set of protocols was executed using iPS cells generated from human dermal fibroblasts by retroviral gene transfer of OCT3, SOX2, and KIF4 and successfully demonstrated their differentiation into retinal cells. The RhoK inhibitor 36 was used in the culture medium to prevent dissociation-associated cell death. Other workers have also described the production of RPE cells from iPS cells using mixtures of transcription factors such as Oct4, Sox2, Nanog, and Lin28.145 Studies using both mouse and human iPS cells were described and produced cells positive for markers of retinal progenitor cells as well as photoreceptors.146 The differentiation of intraocular stem-like cells has also been investigated in vitro. For example, 1 has been described as promoting the in vitro proliferation of rabbit limbal epithelial cells (RLECs), with the mechanism being proposed as improving the expansion of limbal stem-like cells.147 Specifically, concentrations of up to 200 nM of 1 were found to enhance the proliferation of RLECs significantly, although higher concentrations (400 and 800 nM) appeared to be moderately to very toxic. Furthermore, 200 nM concentrations of 1 were found to double the colony forming efficient of RLECs (26% vs 13% in controls). 3.3.3. In Vivo Studies. Direct in vivo modulation of endogenous progenitor and stem cells is the emerging and alternative approach. While there have been relatively few reports to date of small molecule modulation of retinal progenitor cells, either in vitro or in vivo, with the emphasis being instead primarily focused on cell-based therapies, there is increasing evidence of a shift toward the use of small molecules, with examples now being published of studies using some of the better characterized, more promiscuous stem cell modulator compounds (discussed below). Furthermore, as more detailed investigations are carried out regarding the key signaling pathways involved in directing retinal stem, or progenitor cell fate, the discovery and study of small molecules which

have initiated clinical trials in recent years and have been reviewed elsewhere.142 3.3.2. Small Molecule Modulators of Retinal Differentiation. The ultimate aim of this type of study would be the identification of small molecules able to repair damaged cells in vivo following intraocular injection, and a range of strategies have been used by various workers. As a route to understanding how this might be brought about, it is useful to analyze the progress that has been made in differentiating human ES and iPS cells and limbal stem-like cells into RPE cells because such compounds should identify signaling pathways used to produce these cells, suggest new targets for future drug discovery, and in themselves produce a ready supply of cells for in vitro model studies and future drug screening. Following several studies showing the effects of 63 (nicotinamide) on cell differentiation and survival, Reubinoff and co-workers studied how this compound promotes the differentiation of human ES cells to neural cells and from there to RPE cells (Figure 9).143 In an effort to establish in more detail how this process was taking place, genomic analysis of hES cells was undertaken. Results obtained following four weeks of treatment with 63 showed that a number of differentiation pathways, including those relating to hematopoesis and melanogenesis, were significantly enhanced. Furthermore, neural, retinal, and RPE genes were also upregulated, which then served as a justification for more detailed evaluation. In a longer term study, over 70% of cell clusters exhibited pigmented areas following eight weeks of treatment with nicotinamide, and of these clusters around 5% of cells were pigmented and expressed a marker, microphthalmia-associated transcription factor (MITF) present in RPE cells. Importantly, these human ESC-derived RPE cells were shown to be viable in vivo following transplantation into dystrophic RCS rat eyes and show functional and structural rescue six weeks after implantation. The use of casein kinase inhibitor 64 (CKI-7), 33, and RhoK inhibitor 36 has been described as a combination capable of producing retinal precursor cells from hES cells.144 The proteins Dkk1 and Lefty-A have also been shown to cause differentiation of hES cells into retinal precursors, thought to be S



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photoreceptors153 and lower luminescent threshold than untreated animals, suggesting greater photoreceptor preservation. Interestingly, this same diaryl urea derivative 69 was originally described by the same workers as being able to stimulate the proliferation of β-cells (see section 4.6.3). Target identification using an affinity-based strategy similar to that described previously by the same authors identified the same target protein as in β-cells, EBP1.154 Given the observation of proliferative activity in more than one primary cell line, it would be of great interest to see more detailed studies on this compound including mode of action and other cell types affected. While there have been a few studies to date describing the effects of small molecules on retinal differentiation in vitro, there have not, to our knowledge, been any studies investigating the direct administration of such compound in vivo. Nonetheless, because the eye is to a significant extent a self-contained system, it would be of real interest to observe the effects of some of the in vitro active compounds upon direct intraocular administration. 3.4. Chemical Manipulation to Effect Cardiac Regeneration. 3.4.1. Cardiovascular Disease. Cardiovascular disease encompasses a range of conditions including coronary and hypertensive heart disease, which in turn lead to acute myocardial infarction and ultimately heart failure. It continues to be the cause of very significant levels of morbidity and mortality and of high cost to health services throughout the world. Much of the disease involves damage to the musculature of the heart, and the search for agents to prevent, alleviate, or repair this damage continues to be a research goal. The prospect of being able to regenerate human heart tissue either in vivo by the controlled differentiation of stem/progenitor cells in situ or ex vivo by subsequent implantation of the generated cardiomyocytes is of great interest and could well lead to a paradigm shift in the treatment of this disease.155−157 Most importantly for the medicinal chemist is the ability to achieve this goal by the use of small molecules presents an important challenge and opportunity. In the search for small molecule agents capable of influencing the generation and relocation of cardiomyocytes in vivo, significant encouragement can be taken from the work carried out in the area of stem cell transplantation. While reliable clinical measures of efficacy are still being debated and established, methods are being reported for the generation of cardiomyocytes in vitro158 and success in these areas would potentially pave the way for in vivo treatments. For the medicinal chemist, an approach to the search for a small molecule that promotes cardiomyogenesis would be greatly facilitated by an in-depth understanding of the progenitor cells to be used and the signaling pathways involved in the stepwise differentiation of these cells to cardiomyocytes. Jung et al. in an excellent review diagrammatically summarized both the relatively wide range of cells that can be used as progenitors of cardiomyocytes and the small molecules that have been used to induce differentiation to cardiomyocytes and the preparation of cells for the treatment of heart disease.159 Cells ranging from embryonic stem cells and cardiac progenitor cells through to bone marrow stromal cells, peripheral blood mononuclear cells, circulating stem cells, skeletal muscle cells, and hair bulge cells have all been used. In a very recent review, Xie et al. have presented a perspective on heart repair involving cell

modulate these pathways can be expected to follow. For example, disruption of TGFβ signaling in retinal/eye disorders was described in the literature during 2005.148 Development of various ocular cells from the neural crest was reportedly influenced by TGFβ signaling because disruption of this pathway was found to result in downregulation of two transcription factors known to be important for development of the eye (Pitx2 and Foxc1), coupled with ocular defects. Given numerous disclosures in the patent literature since that date of the effect and therapeutic potential of TGFβ modulators in other therapeutic areas,149 it would be of interest to see whether small molecule modulators of this pathway recapitulate these effects. 15 has been shown to promote photoreceptor differentiation in various vertebrate retinal studies, including zebrafish and human, although the precise mechanism by which it does so has yet to be elucidated.150 A recent publication has described the reprogramming of human primary neonatal epidermal keratinocytes to human iPSCs and their directed differentiation to RPE cells using a combination of small molecules and growth factors and their subsequent application in a preclinical model. These included the so-called “2F” conditions, consisting of either Oct4/Klf4 and 6 and 8 or Oct4/Klf4 and 6, 33, 8, and 65. The single growth factor mixture “1F” was also utilized which consisted of Oct4 along with 65 (PD0325901), 66 (sodium butyrate), 67 (PS48), and 68 (A83-01).151 The resulting cells were found to express the RPE terminal differentiation markers bestrophin, CRALBP, and RPE65. Metabolic and proteomic analysis of the 1F and 2F derived RPE cells comparing them to human fetal RPE cells found there to be few differences. In vivo transplantation of 1Fderived RPE cells into the retinal incompetent RCS rat model also demonstrated striking results in that the cells implanted as a correctly polarized and extended monolayer (despite the bolus nature of the injection), with preliminary evidence of electrical responses to light stimulation, suggesting that they were also functionally competent. This approach could therefore be viewed as offering significant promise for the treatment of retinal diseases. Recent work published by Swoboda et al. has demonstrated the effective ex vivo proliferation of RPE cells using the druglike small molecule 69 (WS3).152 The doubling rate of untreated RPE cells was found to have diminished significantly by the sixth passage, whereas those treated with 25 nM of 69 were still doubling at almost maximal levels at passage 15. Because the intended application of this type of ex vivo expansion technology is the provision of cells for applications such as transplantation therapy, assessment of the safety of the product cells is vital. In preliminary experiments to this end, cytogenetic analysis of the resulting cells did not demonstrate any genetic alterations in the resulting cells nor were any tumorigenic events noted. Furthermore, the later passage cells remained functionally competent because when compound 69 was removed (passage 10) and the cells cultured for five weeks, they became pigmented and adopted a cobblestone-type morphology, behavior typical of differentiated RPE cells. RT-PCR analysis of the cells also demonstrated the presence of markers characteristic of mature RPE cells, including RPE65, RAX, MITF, and others. In in vivo studies using the well established RCS (Royal College of Surgeons) rat model, implantation of 69 expanded RPE cells, resulting in a retina with both more T



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Figure 10. Maturation of mESCs into cardiomyocytes. Brach, Gsc stage specific genetic markers; Nkx2.5, homeodomain transcription factor; Mef2c, MADS box transcription factor; αMHC, α-myosin heavy chain; cTNT, cardiac isoform of troponin-T.

Figure 11. Examples of small molecules reported to manipulate cardiac cells in vitro and in vivo.

cytes, thus producing a large supply of cardiomyocytes for cellbased therapeutics.162 Notwithstanding the need for further work to confirm the pathways involved in the conversion of stem cells into cardiomyocytes, several publications have reported the effects of small molecules on this process. 3.4.2. Chemical Manipulation of Cardiac Differentiation. 3.4.2.1. In Vitro Model Studies. As early as 2004, Wu et al. reported a series of small molecules (Cardiogenol A−D), which selectively and efficiently induced murine P19 embryonal carcinoma cells to differentiate into cardiomyocytes, noting that their use would serve as probes for the study of cardiac muscle differentiation and could lead eventually to therapy in the longer term.25 Minami et al. reported the production of clinical grade cardiac cells from human iPSCs using a small molecule 70 (KY02111).163 Although the precise target was not known,

reprogramming in which small molecule approaches may prove useful.160 Although the signaling pathways for the generation of cardiomyocytes are not yet totally clear, a summary of the sequence of events in the transformation of mESC through mesoderm induction, mesoderm patterning, cardiac specification, and finally cardiomyocyte maturation has been presented.161 In the same paper, the authors, very usefully for the medicinal chemist seeking to control each step by the use of a small molecule, illustrate the control of each step by a specific growth factor and show how some of these factors have been applied to mESCs at specific time points during the differentiation process (Figure 10). On the basis of the same differentiation sequence, Parsons et al. reported the use of 63 to induce the differentiation of human ESCs to cardiomesoderm which progressed to cardioblasts and ultimately cardiomyoU



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recently, several studies have suggested that epicardial derived cells (EPDCs) can contribute to cardiac repair in vivo following injury. In a pioneering study by Smart et al. in 2011, it was demonstrated in a murine model that resident adult EPDCs treated with thymosin β4 undergo migration and differentiation into what appear to be structurally coupled cardiomyocytes in situ following injury.174 Thymosin β4 is currently undergoing clinical evaluation for wound healing, corneal repair, and cardiac repair following myocardial infarction. In a pioneering study demonstrating proof-of-principle in this area, a series of 3,5-disubstitued isoxazoles (e.g., 57, 74) have been shown to act in vivo on Notch activated adult epicardium-derived cells (NECs) to generate Notch-activated adult cardiomyocyte precursors.175,176 Although these isoxazoles failed to affect functional parameters after myocardial infarction, nonetheless they represent significant “lead” compounds in the search for more potent and functionally effective inhibitors. These isoxazoles were originally identified via an in vitro phenotypic screen, and in a follow-on paper the same group subsequently identified GPR68 as the target for these molecules, a previously unidentified regulator of myocardial cellular response and thus providing a new approach to targetbased drug discovery in this area.176 In another example of the use of a phenotypic assay in zebrafish embryos, Ni et al. using a readout of increased heart size to identify a series of three related compounds, 74, 75, and 76 (Cardinogen 1−3), which induce cardiomyogenesis. These were subsequently shown to act by inhibiting WNT/β-catenindependent transcription in ES cells and zebrafish embryos.177 Pasha et al. studied the reprogramming of skeletal myoblasts using a DNA methyltransferase inhibitor 78 (RG108) to generate iPS cells, cardiac progenitors from which propagated in the infarcted myocardium without tumorogenesis and with improved cardiac function.178 Graichen et al. reported the use of 79 (SB203580), a p38 MAP kinase inhibitor, in the presence of factors released from the END2 cells to promote the differentiation of human ESC into cardiomyocytes with promising efficiency.179 Furthermore, in a separate study, the same p38 MAPK inhibitor in combination with FGF1 was shown to induce mitosis in cardiomyocytes in vitro and, most importantly, to reduce scarring and improve cardiac function in vivo in rats following acute myocardial infarction.180 Interestingly, treatment of 79 in the absence of FGF1 following acute MI did not lead to a restoration of cardiac function, leading the authors to postulate that increased angiogenesis and/or increased cardiomyocyte survival, attributed to FGF1 treatment, was important for functional recovery. In an effort to test the correlation between such inhibition of MAP kinase and differentiation of human ESCs to cardiomyocytes, Low et al. designed and prepared a series of imidazole analogues and tested them for their ability to both inhibit MAPK and to induce cardiomyocyte differentiation.181 Although they demonstrated that analogues of 79 could induce cardiomyocyte differentiation from ES cells, the correlation between this and MAPK inhibition could not be established and that further work in this area was needed. While the above examples do not represent a comprehensive coverage of the work being carried out in the area of chemical approaches to the manipulation of stem and progenitor cells for the treatment of cardiovascular disease, they nonetheless illustrate the recent advances made in this area. Key proof-ofprinciple studies have been described in animal models and,

the results suggested that the molecule was acting via inhibition of the WNT signaling pathway. They reported that the combined use of 70 and WNT signaling modulators in the absence of recombinant hormones and cytokines resulted in cardiac differentiation, suggesting that this was an efficient means of producing human cardiomyocytes for regeneration therapies (Figure 11). In a mechanistic review, Willems et al. report the use of ESCs and iPSCs to generate cardiomyocytes and to facilitate the elucidation of the biology of stem cells in the human heart.164 Following this work, the initial discovery of a series of dihydropyridines, which induced cardiogenesis, and the medicinal chemistry leading to their identification as inhibitors of TGFβ/Smad signaling by an unprecedented mode of action, is described in a paper by Schade et al.165 They also reported the use of these molecules to identify signals controlling stem cell differentiation to cardiomyocytes and established that 71 (ITD-1) was a selective TGFβ inhibitor, acting by clearing of TGKBR-2 receptor from the cell surface, suggesting a role for this pathway not only in the control of differentiation to cardiomyocytes but in other pathological processes involving TGFβ signaling.166 Ao et al. reported the use of a dorsomorphin homologue, 39, a second-generation BMP inhibitor to induce beating cardiomyocytes from mESCs.167 Compound 39 was shown to enrich procardiac progenitor cells that then respond to WNT inhibition using the known inhibitor, 3,5,7,8-tetrahydro2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin4-one (XAV939), to produce secondary beating cardiomyocytes.167 Initial studies on the use of dorsomorphin to induce myocardial differentiation in mESCs were reported by Hao et al. in 2008.168 Oh et al. used a high-throughput screen of a small molecule peptidomimetic small molecule library to find 72 (CW209E).169 This molecule increased the ratio of beating embryoid bodies without inducing cytotoxicity in mESCs and as such should be useful in identification of targets and in the generation of cardiomyocytes for therapy. Using small molecules including 73 (IWR-1), Ren et al. showed that the modulation of the bone morphogenic protein4 (BMP-4) and the Wnt/β signaling pathways could be exploited to induce efficient cardiac differentiation from human ESCs and iPSCs.170 Similarly, Willems et al. showed that 73 also drives human mesoderm cells to produce cardiomyocytes.171 In other studies, the identification of sulfonyl hydrazones as small molecules involved in cardiac fate was established by screening a chemical library for activators of the Nkx2.5 gene using a luciferase assay in mouse iPSCs.172 Of 33 small molecules evaluated, verapamil and cyclosporin showed the most significant positive effect of cardiomyogenesis in inducing the differentiation of ESCs when tested against an ES cell line transfected with EGFP under the control of a α-myosin promoter. Sachinidis et al. subsequently used this data to contribute to the understanding of the signaling pathways involved in cardiomyogenesis.173 3.4.2.2. Small Molecule in Vivo Manipulation of Cardiac Cells. While a number of cellular in vitro model studies have been reported using small molecule modulators of cardiac differentiation, translation to achieve efficacy in vivo has proved challenging. This may in part have been due to a lack of clarity in field as to which cell types within the adult heart are relevant to target to effect cardiac regeneration in vivo. However, more V



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Figure 12. Examples of small molecules reported to manipulate muscle cells in vitro and in vivo.

(lysophosphatidic acid), and adenylyl cyclase inhibitor 82 (SQ22536) (Figure 12). A study by Ryan et al. demonstrated both increased progenitor numbers and increased skeletal myogenesis following multiday treatment of human ESC with the pleiotropic stem cell modulator 11.184 Enhancement of the expression of the myogenic regulatory factor MyoD was also seen. A mitochondria targeted fluorophore has also been described as being able to influence muscle cell fate.185 While the authors initially intended to undertake a screen to identify compounds which were capable of distinguishing between the various states of muscle differentiation, they identified 83, a rosamine derivative (B25) which reverts myotubes to a mononuclear myoblast phenotype. Following early studies from Schultz et al., who reported myoseverine to be capable of effecting cytoskeletal remodelling to revert myotubes to a myoblast phenotype,186 more recent work has shown phosphatase and apoptosis inhibitors 84 (BpV(phen)) and 85 (Q-VD-OPh), respectively, used in combination as being able to dedifferentiate myotubes to progenitor myogenic cells in the absence of any additional gene overexpression.187 Of particular note with this work was the high efficiency of the process (12%) and that the resulting muscle cells were functionally competent and capable of both self-renewal and redifferentiation. The latter was demonstrated both in vitro and in vivo in SCID mice. In studies from other researchers, the same inorganic chemical 84, a potent inhibitor of phosphotyrosine phosphatase, has been found to dedifferentiate myogenic cells ̈ circulating phenotype.188 These studies further to a more naive complement the 2011 work of Paliwal et al. and have potentially important ramifications for muscle regeneration as

importantly, recent years have seen emerging evidence of tractable populations of cells within the adult heart capable of contributing to regeneration processes. 3.5. Chemical Manipulation of Muscle Regeneration. Degeneration and/or loss of the various types of muscle fibers and tone are associated with a range of disorders, from cardiac disease (e.g., ischemic heart disease) and cachexia (muscle wasting associated with sufferers of cancer, COPD, AIDS, etc.) through to less common genetic diseases such as Duchenne and Becker muscular dystrophies (DMD and BMD). Accordingly, therapeutic approaches such as stem cell modulation which can directly treat (i.e., reverse) the underlying process of muscle loss would be an attractive therapeutic option. A comprehensive review has recently been published, describing a wide range of compounds which repair or replace defective muscle tissue. Notably though there are currently no drugs targeting muscle stem cells which have been approved for that specific purpose, and interestingly there are also no muscle focused cell-based therapeutic agents on the market as yet. However, as with other areas of regenerative medicine, there is an active and growing body of research into agents which can affect muscle, with a considerable number of examples have already been described. In this emerging stem cell-based therapeutic approach to muscle disorders, as might be expected, initial publications have focused very much on the relatively small (but growing) number of well characterized but promiscuous small molecules which have been described as being able to modulate stem cell fate in other target tissues. For example, a number of small molecules have been described as inducing dedifferentiation in myotubes.182,183 These include GSK3β inhibitor 80 (BIO), p38 MAPK inhibitor 74, broad spectrum GPCR activator 81 W



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Figure 13. Examples of small molecules reported to manipulate pancreatic cells in vitro or in vivo.

reviewed, inter alia, by Baetge.194 Other studies192,193 found that 88 ((−)-indolactam V) transformed endoderm cells derived from human embryonic stem cells into Pdx1-expressing progenitor cells (Figure 13).195,196 Pdx1 is pancreatic and duodenum homeobox 1, a transcription factor which is necessary for pancreatic development.192 Baetge described how the differentiation of a stem cell to a βcell is a multistep process and that it is essential to rigorously identify each cell type in the process to ensure the integrity of the final product.194 A crucial step is to use 11 in conjunction with Sonic Hedgehog modulation to commit the differentiation toward pancreatic lineage and to skew differentiation of the pancreatic endoderm to endocrinal cells rather than acinar cells. It has further been reported that pancreatic progenitor cells generated from embryonic stem cells when grafted into mice can mature into cells that have much of the functionality of βcells.197 4.6.3. In Vivo Stimulation of β-Cells. While a degree of control of diabetes could be achieved by transplantation of new β-cells, a better method would be to trigger the renewal process in vivo by dosing with agents able to provoke the transformation of existing cells into β-cells. A crucial element in this approach is the finding that the adult pancreas shows a surprising degree of plasticity and potential for regeneration following injury. Further, the existence, or otherwise, of pancreatic stem cells in adults was further discussed by Lee et al.198 While pancreatic duct cells retain the ability to expand and differentiate and can reverse hyperglycaemia on transplantation, it does appear that β-cell regeneration takes place mainly from existing β-cells and that terminally differentiated βcells retain proliferative capacity. It is obvious that in trying to design a therapeutic approach based on stimulating existing cells to replace damaged β-cells it has to be shown exactly which cells are responsible for observed proliferation. Pancreatic stem/progenitor cells were isolated from the rabbit and shown to be able to proliferate strongly and to secrete insulin on stimulation by high glucose concentrations.199 A number of groups have been studying the zebrafish and have shown significant similarities between its pancreatic development and that of mammals200 and shown that it is possible to do high-throughput screening in this species, thus identifying molecules able to affect the differentiation process to β-cells. The use of zebrafish embryos was reported by Andersson et al.,201 who used zebrafish embryos genetically modified to express a fusion protein of cyan fluorescent protein (CFP) and nitroreductase under the control of the insulin promotor. The purpose of the nitroreductase enzyme was to produce toxic metabolites from metranidazole and thus damage the pancreas, allowing temporary controlled β-cell ablation. A high-throughput screen then tested 7186 compounds and found five which would regenerate the β-cells. Of these, four

the 84-treated cells were observed to reach muscle and contribute to repair following systemic administration.187 The type 1 TGFβ inhibitor 86 (CalBio 616452) has been shown to restore regenerative function to stem cells in aged muscle. This provides further evidence that modulation on this signaling pathway could play an important role in regenerative potential for muscle.189 Natural product screening has also provided some interesting preliminary results. In studies of the biological activity of extracts from Geum japonicum, a pure fraction was found to be active and to stimulate differentiation of myogenic precursor cells, leading to regeneration of myofibers and giving more efficient muscle repair.190 The structure of the active principal was elucidated using NMR studies and found to correspond to a previously described triterpenoid 87 (“Trit”).191 3.6. Chemical Manipulation of Pancreatic Regeneration. 3.6.1. The Pancreas, β-Cells, and Diabetes Mellitus. βCells, which comprise less than 1% of the total cells of the pancreas, release insulin, the hormone responsible for controlling blood glucose levels, and damage to these cells results in the disease diabetes mellitus. Type I diabetes results from an autoimmune attack on β-cells and requires life-long daily subcutaneous injection of insulin to sustain life. While this treatment is highly effective, it cannot accurately reproduce the normal functioning of β-cells with their glucose sensor, the rapid production of insulin, the release of this insulin in response to glucose, and the maintenance of normoglycemia essentially indefinitely. Because of the lack of exquisite control of insulin levels, over long periods of dosing there can arise complications such as diabetic retinopathy, nephropathy, heart disease, and stroke. Thus, there has been considerable interest in finding treatments which restore the normal functioning of the β-cells. Among potential treatments are the generation of βcells from stem cells either ex vivo followed by transplantation or in vivo. The formation of the different pancreatic cell types in the embryo has been discussed,192 and these clearly show the stepwise nature of the maturation process. It is also clear that cell−cell interactions, particularly with endothelial cells, affect the maturation, and β-cells interact strongly with each other through ephrins, cell adhesion molecules, and gap junctions. Very recently, Narayanan and colleagues have shown that human ESCs can be induced to give insulin producing β-cells by interaction with decellularized extracellular matrix and conditioned media from the appropriate committed cell lines. The resultant cells were able to produce insulin in diabetic mice although teratomas were also produced, indicating that further purification of the β-cells was required.193 3.6.2. In Vitro Pancreatic Cell Differentiation. The differentiation of human ESCs or iPSCs into β-cells has been pursued by a number of groups, and their efforts have been X



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effects in other tissues. Moreover, there may be concerns that candidate molecules may inappropriately activate stem cell populations even within the target tissue of interest, leading to disorders such as oncogenesis and fibrosis or even possible depletion of the endogenous stem cell pool. It is also likely, therefore, that the in vivo exposure of stem cell activating molecules will need to be carefully tuned as compounds are progressed. Nonetheless, there are now a growing number of examples demonstrating that this specificity is achievable in vivo. Specificity and appropriate potency have long been criteria that medicinal chemists work to however, and as such these challenges cannot be seen as either overwhelming or insurmountable. Furthermore, with advances in iPS cell technology and an increased understanding of the stem cell niche, more robust and reliable in vitro screening tools are likely to become available which should facilitate this important issue. The eventual therapeutic use of “hit” molecules, discovered in the laboratory and characterized in the laboratory largely using in vitro technologies, will equally depend upon the successful resolution of all the properties, such as ADMET, offtarget idiosyncratic activity, in vivo animal model relevance, synthetic availability, stability, etc. with which medicinal chemists undertaking “hit” to “lead” studies are all too familiar. An early assessment of the likelihood of “hit” or “lead” compounds translating to a clinical setting is also of paramount importance, and human iPS cell technology will undoubtedly continue to facilitate the production of human cells and tissues as models for analysis. The rewards, however, should successful molecules be developed, promise to be spectacular. We have already seen the contribution made by eltrombopag in this area, and significant advances have been made in many other disease states. The many in vivo proof-of-principle experiments reported in a range of tissue types demonstrates the feasibility of the small molecule approach. This, alongside improvements in in vitro screening tools, methods, and biomarkers to monitor efficacy in vivo and our emerging understanding of stem cells and their niches, means we are poised for the discovery and development of a new generation of regenerative medicines. The discovery of small molecules for the treatment of serious diseases such as neuronal degeneration, cardiac failure, diabetes, and many other diseases for which there are no effective treatments is not an unreasonable goal, which we hope this perspective will help to stimulate.

compounds are involved in the adenosine signaling pathway and the most active was 89 (NECA, 5-N-ethylcarboxamidoadenosine). Importantly, the effects were demonstrated to be specific to β-cells, not other endocrine cells or cells within other tissues, and they went on to demonstrate that 89 also stimulated β-cell proliferation ex vivo in isolated murine islets and in vivo in a diabetic mouse model, suggesting adenosine signaling is an evolutionarily conserved mechanism for β-cell regeneration. Adenosine receptor A2a signaling was later confirmed as the pathway through which NECA exerted its effects, suggesting possible molecular targets for potential future drug development. Recently, Schultz et al. have reported the discovery of a series of diarylureas and amides including 90 (WS6) as inducers of βcell proliferation in vitro and in vivo.154 These compounds were originally discovered in an in vitro high-throughput screen using a rodent β-cell line. Excitingly, 90 was subsequently shown to be active in primary rat and human islets, to have a promising pharmacokinetic profile, and to stimulate β-cell proliferation and lower glucose levels in vivo in a murine model of type I diabetes. Affinity-based target pull-down studies identified two proteins, Erb3 binding protein-1 (EBP-1) and IKKε, as potential targets. Interestingly while knockdown of EBP-1 alone also induced β-cell proliferation, IKKε inhibitor treatment alone did not. Further, knockdown of EBP-1 in combination with either 90 or IKKε inhibitor showed similar levels of activity to treatment with 90 alone, suggesting a synergistic role for these two targets in β-cell proliferation. This latter result again serves to underline the immense value a phenotypic screening strategy can offer as opposed to a targetcentric screening approach. The pancreas is possibly the organ in which most research has already been done to stimulate the growth of one cell type, namely the β-cell, and the results described above give considerable optimism that the goal of finding a drug able to achieve this could be realized.

4. CHALLENGES AND CONCLUSIONS The purpose of this perspective is to stimulate the medicinal chemist to become concerned with the application of their extensive skills and experience to stemistry,1 the discovery of small molecules that stimulate the proliferation and differentiation of endogenous stem cells within damaged tissue to effect the repair of that tissue. Although significant and ever accelerating progress is being made in this field, as exemplified by examples cited in this perspective, nonetheless significant challenges remain. These challenges are, however, familiar to the practicing medicinal chemist, and even if perhaps more exaggerated in this area than in traditional receptor enzyme modulation, they are challenges which can be optimistically faced. The search for “hit” molecules in areas where mechanistic pathways are less than fully established is being overcome by more efficient phenotypic screening, with the “hits” themselves being used to unravel mechanistic pathways and hence facilitate further progress by high-throughput target screening and subsequent optimization of the resulting compounds. As more information about the key intracellular signaling pathways is gained, the scope to use structure-based drug design will also grow. The need for exquisite specificity such that the stem cell activation is restricted to the target organ is likely to be paramount, particularly where inappropriate or excessive stimulation can lead to the formation of tumors or undesired

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AUTHOR INFORMATION

Corresponding Authors

*For A.J.R.: phone, +44(0)1865 275643; E-mail, angela. [email protected]. *For S.G.D.: phone, +44(0)1865 275646; fax, +44(0)1865 285002; E-mail, [email protected]. Notes

The authors declare the following competing financial interest(s): A.J.R. and S.G.D are founders of a University of Oxford spin-out company, OxStem Ltd. P.D.K., P.T.S., R.W. and G.M.W. declare no competing financial interests. Biographies Stephen G. Davies received his B.A. in 1973 and D.Phil. in 1975 from the University of Oxford. After a stint at the CNRS at Gif-sur-Yvette, he returned to Oxford in 1980 to a University Lectureship and then a Professorship. In February 2006, he was appointed as the Waynflete Y



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progressed into clinical trials. In 2005, he joined the University of Oxford spin-out company VASTOx Ltd (now Summit plc), leading the medicinal chemistry team which discovered SMT-C1100, a potential first-in-class treatment for Duchenne muscular dystrophy. Since 2009, he has worked at the University of Oxford with Dr. Angela Russell and Professor Steve Davies on various drug discovery targets, including oncology, muscular dystrophy, and stem cell modulation.

Professor of Organic Chemistry. He has published >500 research papers encompassing a huge array of topics varying from organometallic chemistry and asymmetric synthesis to medicinal chemistry. He has a number of multidisciplinary research collaborations that include the development of antitubercular and anticancer agents, a novel therapy for Duchenne muscular dystrophy, and small molecules to manipulate stem cell fate. He has founded several successful companies including Oxford Asymmetry (now Evotec) and VastOx (now Summit plc).

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ACKNOWLEDGMENTS A.J.R. and S.G.D. thank the EU, the British Heart Foundation Centre of Research Excellence in Oxford, and Shionogi Inc. for funding their laboratory’s work on stem cell chemistry. A.J.R. also thanks Research Councils’ UK for a Fellowship. P.T.S. thanks New College, Oxford, for a Junior Research Fellowship.

Peter D. Kennewell studied Natural Sciences at the University of Cambridge and gained his Ph.D. with Professor Alan Katritzky at the University of East Anglia. He was a postdoctoral fellow with Professor Ned Heindel at Lehigh University, Bethlehem, Pennsylvania, working on antimalarial agents. He joined Roussel Laboratories (now part of Sanofi-Aventis) as a medicinal chemist and subsequently held a number of increasingly senior research management roles until the UK research department was closed following the merger of HoechstRoussel with Marion Merrell Dow in 1995. He then joined the Biotechnology and Biological Sciences Research Council with a particular interest in encouraging chemists to apply their skills to attacking biological problems. In retirement, he has mentored a number of on-line medicinal chemistry courses.

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ABBREVIATIONS USED AD, Alzheimer’s disease; AhR, aryl hydrocarbon receptor; AKT, protein kinase B; ALP, alkaline phosphatase; ALS, amyotrophic lateral sclerosis; AMD, age-related macular degeneration; BMMSC, bone-marrow derived mesenchymal stem cell; BMP, bone morphogenetic protein; BMP-4, bone morphogenetic protein-4; CREB, cAMP response element-binding protein; CK1, casein kinase 1; CXCR-4, C-X-C chemokine receptor type 4; DR, diabetic retinopathy; EGF, epidermal growth factor; EPO, erythropoietin; ERK, extracellular signal-related kinase; ESC, embryonic stem cell; FAK, focal adhesion kinase; FGF, fibroblast growth factor; GAP, guanosine triphosphatase activating protein; G-CSF, granulocyte colony stimulating factor; GFP, green fluorescent protein; GM-CSF, granulocyte macrophage colony-stimulating factor; GSK-3β, glycogen synthase kinase-3β; GvHD, graft-versus-host disease; HDACi, histone deacetylase inhibitors; HGK, mitogen-activated protein kinase kinase kinase kinase 4; HIF-1α, hypoxia-inducible factor1α; HSC, hematopoietic stem cells; HTS, high-throughput screening; IGF-1, insulin-like growth factor-1; iPSC, induced pluripotent stem cells; LIF, leukemia inhibitory factor; MEP, megakaryocyte/erythrocyte progenitor; MLCK, myosin light chain kinase; MPL, myeloproliferative leukemia protein; MSC, mesenchymal mutlipotent stromal cell/mesenchymal stem cell; NGF, nerve growth factor; NOD/SCID mouse, nonobese diabetic/severe combined immunodeficiency mouse; NPC, neural progenitor cell; NSC, neural stem cell; PDGFR, platelet-derived growth factor receptor; PI3K, phosphatidinylinositol 3-kinase; RCS, retinal stem cells; RLEC, rabbit limbal epithelial cells; RMS, rostral migratory stream; RPE, retinal pigment epithelium; SCF, stem cell factor; SDF-1, stromal cellderived factor-1; smNPCs, small molecule neural precursor cells; SVZ, subventricular zone; TBI, traumatic brain injury; TrkA, tyrosine kinase receptor type A; TGFβ, transforming growth factor-β; TPO, thrombopoietin; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor; VLA4, very late antigen-4

Angela J. Russell gained her M.Chem. degree from the University of Oxford in 2000 and her D.Phil. in Organic Chemistry in 2004 under the joint supervision of Professor Steve Davies and Dr. Tim Perera (Yamanouchi plc). In July 2007, she was awarded a Research Councils’ UK Fellowship in Medicinal Chemistry jointly between the Departments of Chemistry and Pharmacology in Oxford and in 2012 was appointed as a University Lecturer in Medicinal Chemistry. Her research interests encompass medicinal chemistry and drug discovery and include the development of anticancer agents, a new therapy for Duchenne muscular dystrophy, signaling pathway modulators, and small molecules to manipulate stem cell fate. Peter T. Seden obtained his first degree in Natural Sciences from the University of Cambridge (Selwyn College) before moving to Bristol to complete a Ph.D. in natural product synthesis under the supervision of Professor Chris Willis. He then moved to the University of Oxford to undertake postdoctoral research in the field of medicinal chemistry, working on projects funded by Cancer Research UK under the supervision of Professor Steve Davies, Professor Chris Schofield, and Dr. Angela Russell. In 2011, he became a Junior Research Fellow of New College Oxford, investigating the use of small molecules to control stem cell fate. He now works as a chemist within the Formulated Products Technology Division of BP. Robert Westwood studied Chemistry at the University of Hull and gained his Ph.D. with Dr. R. M. Scrowston and Professor N. B. Chapman. He was a postdoctoral fellow with Professor J. W. Lown in the University of Alberta, Edmonton, and with Professor D. H. Williams in the University of Cambridge. In the industrial environment, he was Research Director for Roussel Laboratories Ltd in the UK before joining Roussel Uclaf in Paris as Responsable Recherche Immunology. After a brief period as Professor of Medicinal Chemistry at Hull University, he then joined Cyclacel Ltd. as Head of Preclinical Development. He is currently a visiting academic at the University of Oxford.

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AF


Regenerative medicinal chemistry: the in situ control of stem cells.

Probing the binding site of abl tyrosine kinase using in situ click chemistry.

In situ freezing of embryonic stem cells in multiwell plates.

TGF-β signaling in the control of hematopoietic stem cells.

In Situ Quantification of Surface Chemistry in Porous Collagen Biomaterials.

Quality control in clinical chemistry.

Electrical control of calcium oscillations in mesenchymal stem cells using microsecond pulsed electric fields.

In situ altering of the extracellular matrix to direct the programming of endogenous stem cells.

In situ click chemistry generation of cyclooxygenase-2 inhibitors.

In situ identification of bipotent stem cells in the mammary gland.

In situ characterization of the mTORC1 during adipogenesis of human adult stem cells on chip.

In Situ Recruitment of Human Bone Marrow-Derived Mesenchymal Stem Cells Using Chemokines for Articular Cartilage Regeneration.

MicroRNA control of epithelial-mesenchymal transition in cancer stem cells.

Labeling proteins on live mammalian cells using click chemistry.

Body Management: Mesenchymal Stem Cells Control the Internal Regenerator.

Modeling the tumor microenvironment using chitosan-alginate scaffolds to control the stem-like state of glioblastoma cells.

Stem cells on the brain: modeling neurodevelopmental and neurodegenerative diseases using human induced pluripotent stem cells.

Factors that control the chemistry of the LOV domain photocycle.

Spatial control of adult stem cell fate using nanotopographic cues.

TRES predicts transcription control in embryonic stem cells.

Tumour control probability in cancer stem cells hypothesis.

Quality control: Genome maintenance in pluripotent stem cells.

Markers for the identification of tendon-derived stem cells in vitro and tendon stem cells in situ - update and future development.

Signaling Control of Differentiation of Embryonic Stem Cells toward Mesendoderm.