Reviews pubs.acs.org/acschemicalbiology
Small Molecules Targeting in Vivo Tissue Regeneration Daniel Lan̈ gle,† Jonas Halver,† Bernd Rathmer,† Erik Willems,‡ and Dennis Schade*,† †
Faculty of Chemistry & Chemical Biology, TU Dortmund University, Otto-Hahn-Str. 6, 44227 Dortmund, Germany Muscle Development and Regeneration Program, Sanford-Burnham Medical Research Institute, 10901 North Torrey Pines Road, La Jolla, California 92037, United States
‡
ABSTRACT: The field of regenerative medicine has boomed in recent years thanks to milestone discoveries in stem cell biology and tissue engineering, which has been driving paradigm shifts in the pharmacotherapy of degenerative and ischemic diseases. Small molecule-mediated replenishment of lost and/or dysfunctional tissue in vivo, however, is still in its infancy due to a limited understanding of mechanisms that control such endogenous processes of tissue homeostasis or regeneration. Here, we discuss current progress using small molecules targeting in vivo aspects of regeneration, including adult stem cells, stem cell niches, and mechanisms of homing, mobilization, and engraftment as well as somatic cell proliferation. Many of these compounds derived from both knowledge-based design and screening campaigns, illustrating the feasibility of translating in vitro discovery to in vivo regeneration. These early examples of drug-mediated in vivo regeneration provide a glimpse of the future directions of in vivo regenerative medicine approaches. lthough the number of ″new chemical entities″ (NCEs) and approved drugs has slightly increased in recent years, overall productivity and output of the pharmaceutical industry is constantly decreasing while facing consequences of the demographic change in health care.1−3 Evidently, there is a need for drug candidates with unprecedented modes of action, such as pharmacological agents that treat the cause of a disease and not the symptoms or by restoring cell, tissue or organ function through regenerative mechanisms. It is now important to pursue transformative strategies that will drive paradigm shifts in pharmacological intervention and ultimately provide a true benefit over existing medications. In this regard, the field of regenerative medicine has expanded tremendously in recent years owing to milestone discoveries in stem cell (SC) biology and regeneration. Per definition, regenerative medicine deals with the replacement or regeneration of human somatic cells, tissue, or organs to restore or establish normal function.4 The rapidly evolving field of SC research thus holds great promise for novel therapies of various disorders, addressing unmet medical needs. From the medicinal chemist’s point of view, the development of small molecules that control SC fate is of tremendous interest for various regenerative medicine applications as it opens up the druggable target space. Such applications not only span small molecules as regenerative therapeutics for many distinct diseases but also cover their use for SC-based technologies for safety pharmacology or disease modeling, such as disease-in-a-dish applications.5−7 With regards to therapeutic regeneration, several approaches are being pursued: (1) transplantation of cells that provide regenerative molecules or factors in a paracrine fashion, (2) transplantation of cells that home and engraft into the target
A
© 2013 American Chemical Society
tissue, and (3) conventional pharmacological intervention through direct in vivo delivery of small molecule drugs (or biologicals) that trigger endogenous regeneration (Figure 1).8,9 This review summarizes current progress using small molecules as regenerative agents with a particular focus on compounds that may be directly implemented to treat patients. We describe adult stem and progenitor cells (SPCs), stem cell niches, and mechanisms of homing, mobilization, and engraftment as well as somatic cell proliferation as potential drug targets, with a heavy focus on in vivo applications. Small molecules solely for in vitro maintenance and expansion of pluripotent stem cells or directed differentiation are detailed elsewhere, but the molecules with in vivo relevance are highlighted in this review (see refs 6 and 10−12). General Considerations in the Discovery of (in Vivo) Regenerative Small Molecules. Small molecules have many advantages over proteins or nucleic acid-based agents (i.e., biologicals) for studying SC biology or regenerative therapeutics. They are (physico)chemically well-defined and can be produced in reproducible quality. Moreover, small molecules are (mostly) cell-permeable and can thus selectively modulate intracellular processes and typically do so in a reversible, competitive fashion with their cellular targets. Importantly, they are amenable to medicinal chemistry-driven optimization with regard to their pharmacodynamic (PD) and pharmacokinetic Special Issue: Stem Cell Biology and Regenerative Medicine Received: November 4, 2013 Accepted: December 29, 2013 Published: December 30, 2013 57
dx.doi.org/10.1021/cb4008277 | ACS Chem. Biol. 2014, 9, 57−71
ACS Chemical Biology
Reviews
Figure 1. Overview on possible approaches, strategies, and targets for the identification and development of small molecules for regenerative medicine.
artificial assay looking at the target of interest is screened against millions of compounds. A major limitation of this approach is the focus on the target rather than the disease. This approach hinges on the successes of basic biology studies, where targets are revealed, and for which then in vitro assays can be developed. The recent adoption of high-throughput screening in the academic sector has accelerated drug discovery, where phenotypic disease-relevant readouts have spurred the identification of novel targets. The main difference in these efforts is the use of complex phenotypic readouts, rather than the activity of a single target. This approach allows not only the discovery of novel drug candidates but, likewise important, also the use of functional genomics to rapidly identify novel (druggable) biological targets. These can subsequently be introduced in classical HTS to generate NCEs. Here, we provide examples of small molecules derived from such strategies and illustrate how their in vitro discovery has benefitted the in vivo context.
(PK) properties. Another striking advantage is their lack of immunogenicity compared to many protein therapeutics.5,6,13,14 Fundamental to the identification and development of a potential drug candidate is an in-depth knowledge of (patho)physiological processes to be targeted. A pivotal question is then how to discover small molecules that are suitable as potential in vivo regenerative drug candidates or, at least, as chemical probes for studying in vivo regeneration? As illustrated in Figure 1, endogenous sources for tissue regeneration are tissue-specific (adult) stem and progenitor cells. Such cell populations could be targeted directly to selfrenew and proliferate or to differentiate toward specialized cells within their lineage. Moreover, their mobilization from the niche, migration, and homing to locations of injury and engraftment into the respective tissue could be targeted. Different approaches targeting SPC-dependent regenerative processes could be through modulation of the microenvironment (i.e., stem cell niche) where these SPCs reside. In addition, driving the proliferation of somatic cells represents another means to tissue regeneration. Finally, direct reprogramming of somatic cells is an attractive strategy to regenerate functional tissue. Until now, however, there has been only limited understanding of the signals and mechanisms that control such endogenous processes of tissue homeostasis or repair and regeneration after tissue injury. Knowledge gained from developmental studies with mammalian embryos, as well as animal models of regeneration such as zebrafish or amphibians and from in vitro model systems using induced (or embryonic) pluripotent stem cells, has contributed to our increased understanding of regenerative mechanisms. For example, the hematopoietic stem cell (HSC) system can be seen as a role model for organ regeneration because of extensive research over the past few decades, and knowledge can possibly be extrapolated to other organs.15 Nevertheless, for other organs, the focus should not only be the discovery of small molecules but also the identification of novel druggable targets. In the pharmaceutical industry, drugs are usually identified with a target-centric approach, where an
■
TARGETING ADULT STEM CELLS In the past few decades, mounting evidence for the presence and role of tissue-specific, multipotent adult stem or progenitor cells has sparked different regenerative approaches to treat disease.16 Well-known and characterized examples include hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), neural stem cells (NSCs), skeletal muscle SCs (i.e., satellite cells), or stem cells in the intestinal crypt.17−21 In other therapeutically relevant organs such as the heart and pancreas we are only beginning to understand the biology of adult stem cells.22,23 Given their central roles in tissue homeostasis and regeneration upon injury, adult SCs represent an attractive target for regenerative pharmacotherapy. Interestingly, there are distinct models of the role and state of adult SCs within their habitat, commonly called ‘stem cell niches’, suggesting their existence as both quiescent and active SCs in the same tissue.24,25 Moreover, there are pronounced differences in the regenerative capacity of the various adult SCs. In contrast to 58
dx.doi.org/10.1021/cb4008277 | ACS Chem. Biol. 2014, 9, 57−71
ACS Chemical Biology
Reviews
Figure 2. Pharmaceutical development of the TPO receptor agonist eltrombopag (SB-497115). Key achievements within hit-to-lead and lead optimization are illustrated.
molecule drug that has been brought to the market and developed through a knowledge-driven campaign. In fact, the well-studied biology of hematopoiesis served as a good starting point to its rational development. Since it was known that TPO acts through binding and activation of its receptor, thereby initiating signaling cascades that involve several pathways such as JAK-3, STAT-5, and MAPK, setting up a screening assay that detects TpoR-mediated signal transduction was deemed a straightforward strategy.30 The azo dye SKF-56485 (Orange II) was one of the many hits and further pursued because of its consistent bioactivity, although not exhibiting good “hit-” or “lead-like” features (Figure 2). Thus, comprehensive optimization efforts were undertaken, first tackling a possible in vivo reduction of the azo group by intestinal bacteria as a major concern. This metabolism would not only limit oral bioavailability but also give rise to toxic metabolites. An elegant solution was shifting tautomerism of the azo form to a hydrazone that is resistant to metabolic azoreduction as shown for tartrazine.31 Indeed, replacing the azonaphthol in SKF56485 by a hydrazopyrazolinone and additional SAR investigations furnished the potent and highly effective SB394725.32 The next hurdle was the lack of oral bioavailability. Although this obstacle could be overcome (in rats) by replacing SO3H with COOH in SB-394725, such carboxylic acid analogues were much less potent. However, through molecular modeling and by superimposing the key pharmacophores of SB-394725 and SB-450572 (Figure 2), the investigators realized that the acidic group should be placed at farther distance from the putative metal-chelating motif. On the basis of this information, eltrombopag was designed as an orally bioavailable, highly potent nonsulfonic acid hydrazopyrazolinone carrying a carboxylic acid group on a biphenyl spacer.33,34 In analogy to TPO, various erythropoietin (EPO) analogues are therapeutically used as erythropoiesis-stimulating agents for the treatment of anemia triggered by chemotherapy or renal failure. EPO interacts with its receptor to stimulate proliferation of erythroid progenitor cells and induces their survival and differentiation into red blood cells. To date, however, no small molecule mimic of EPO has been reported. Few EPO peptide agonists have been developed of which one was approved by the FDA in 2012 (i.e., peginesatide).35 However, it was recently recalled because of potentially lethal side effects.36 This underlines the importance of developing small molecule EPO
highly regenerative systems such as the bone marrow (HSCs and MSCs), most organs are endowed with a limited regenerative capacity. Simply said, MSCs and HSCs need to regenerate all life-long, whereas other organs like the heart and kidney should only do so upon injury. A profound understanding of how adult SCs are regulated under physiological conditions, and more importantly upon injury or disease, is essential to the development of small molecule modulators as potential regenerative agents. Here, we discuss (direct) therapeutic targeting of adult SCs fate, namely, their selfrenewal, expansion, proliferation, and differentiation. Hematopoietic Stem Cells. Hematopoietic stem cells (HSCs) are multipotent cells that reside in their niche within the bone marrow and give rise to all cell types of the blood lineage. For over 40 years now, HSC-based transplantation therapies have been widely used in the clinic to replenish cells from the hematopoietic system in order to treat blood disorders of different etiologies. However, a major bottleneck still remains in the difficulty to obtain sufficient HSC numbers as well as immunologically matched HSCs in the case of allogeneic transplantation.20 Therefore, compounds that promote the in vitro or in vivo expansion, proliferation, survival of HSCs or their in vivo mobilization from the niche would aid in overcoming these limitations. Additionally, small molecules that direct differentiation of HSCs toward blood cells are already used for treating certain anemias. For example, thrombopoietin (TPO) is a key regulator of megakaryopoiesis and thrombopoiesis and acts through binding to its cell-surface receptor (TpoR) that is expressed on HSCs, immature hematopoietic cells, and megakaryocytes.26 Since platelets are essential in hemostasis, low platelet counts (i.e., thrombocytopenia) can result in the development of various disorders. TPO and TPO mimetics can be therapeutically used for the treatment of thrombocytopenia of different etiologies such as from chemotherapy, hepatitis C, immune reactions, or as a result of heparin therapy. TPO therapies have been, however, associated with severe complications, i.e., induction of thrombocytopenia because of cross-reacted antibodies against endogenous TPO as an immunogenic response.27 Further development was aimed at second generation non-immunogenic peptides (e.g., romiplostim) and small molecule (e.g., eltrombopag, SB-497115) alternatives.28,29 The latter can eventually be considered the first in vivo regenerative small 59
dx.doi.org/10.1021/cb4008277 | ACS Chem. Biol. 2014, 9, 57−71
ACS Chemical Biology
Reviews
Figure 3. Selected small molecules targeting tissue-specific adult stem cells. Hematopoietic stem cells (HSCs): self-renewal and expansion is promoted by stemregenin1 and eupalinilide E. CASIN generates a rejuvenated phenotype of HSCs. Mesenchymal stem cell (MSC) differentiation toward cartilage tissue is promoted by kartogenin. Cardiac progenitor cells (CPCs) are beneficially stimulated by Isx-1 under homeostasis but not after infarction. Neural stem cell (NSC) expansion is stimulated by antidepressants (reboxetine, fluoxetine, tranylcypromine) and the antidiabetic metformin. NSC differentiation is promoted by neuropathiazol and its optimized analog KHS101. The anticholinergic drug benztropine stimulates oligodendrocyte progenitor cell (OPC) differentiation for multiple sclerosis treatment. P7C3 supports neurogenesis by protection of newborn (from NSCs) neurons.
In addition to ex vivo promotion of HSC expansion, eupalinilide E inhibits differentiation to red blood cells and works in synergy with AhR antagonism underlining a distinct mode of action that has not been identified yet.39 Finally, tackling aging-related mechanisms of cell and tissue regeneration is another appealing strategy, since aged tissues exhibit a lower regenerative capacity compared to younger tissue.40 For example, such aging-related mechanisms have been associated with the small RhoGTPase Cdc42, which plays a key role in cell growth and development. The tricyclic indole CASIN is an inhibitor of Cdc42 and has originally been identified in a phenotypic screen for actin assembly in cytoplasmic extracts (Figure 3).41 Indeed, pharmacological inhibition of Cdc42 by CASIN generates a functionally rejuvenated phenotype of HSCs, revealing novel insight into molecular mechanisms of stem cell aging and at the same time providing a druggable strategy to improve HSC-dependent regenerative processes.42 Mesenchymal Stem Cells. Mesenchymal stem cells mainly reside in the bone marrow but also in adipose or vascular tissue, muscle, and dermis and are an attractive target for pharmacological manipulation.21,43 Particularly for cell-based therapies, MSCs received great attention, primarily due to their therapeutically valuable secretome that exerts various beneficial paracrine signaling roles, but also because of their low or nonimmunogenic nature.44 Among the most interesting small molecules in this field is a chondrogenic compound called kartogenin (Figure 3). Schultz and co-workers identified this molecule from a high-content
mimetics. Such programs are ongoing, such as for example with LG5640 (Ligand Pharmaceuticals) as an attempt to build on successes with TPO mimetics. In addition to small molecules that affect lineage commitment of HSCs in vivo for therapeutic applications, as exemplified with TPO mimetics, chemical modulation of HSC expansion and survival is an attractive strategy to improve the number and quality of cells for transplantation. This is needed for bone marrow-derived HSCs and is even more important for umbilical cord blood-derived HSCs. The latter have emerged as an attractive source of SCs to treat patients suffering from malignant and nonmalignant hematological diseases because of their easy accessibility and since they represent immunologically naı̈ve cells, which increases chances to identify HLA-compatible HSCs.37 Quite a few small molecules improve HSC maintenance and self-renewal in vitro. In particular, compounds that modulate the epigenetic landscape support these processes, such as chlamydocin and trichostatin A (HDAC inhibitors) and 5-AzaC (DNA methyltransferase inhibitor).6 The synthetic adenine derivative stemregenin1 (SR1, Figure 3) promotes the selfrenewal of ex vivo cultured HSCs and has been identified from an image-based screen in primary CD34+ cells. Mechanistic studies revealed that SR1 exerts its activity through antagonism of the aryl hydrocarbon receptor (AhR).38 Along those lines, another interesting small molecule stimulator of megakaryopoiesis is the natural product eupalinilide E, which has similarly been discovered via unbiased high-content screening in CD34+ expressing hematopoietic stem and progenitor cells (Figure 3). 60
dx.doi.org/10.1021/cb4008277 | ACS Chem. Biol. 2014, 9, 57−71
ACS Chemical Biology
Reviews
screen (ca. 22,000 compounds) for chondrogenic nodule formation in primary human MSCs.45 Kartogenin’s chondrogenic profile is attributed to the protection of chondrocytes as well as a direct chondrogenic effect on MSCs within the cartilage. The mechanism of action turned out to be binding to filamin A, which interrupts interaction with CBFβ and prompts its translocation to the nucleus where it affects CBFβ-RUNX1related transcription. Interestingly, efficacy was also demonstrated in an in vivo model of joint injury (mouse). Therefore, kartogenin or lead-optimized derivatives hold great promise as novel therapeutics for cartilage regeneration in degenerative diseases such as osteoarthritis. Thanks to their inherent capacity of differentiation toward osteoblasts, direct stimulation of MSC populations is also attractive for the treatment of osteoporosis from diverse etiologies (i.e., through bone regeneration). For instance, the adenine derivative purmorphamine was discovered from a screen of 50,000 heterocyclic compounds in mouse embryonic mesoderm fibroblasts (i.e., mesenchymal progenitors) as a model system for osteoblast differentiation and was characterized as an inducer of osteoblast formation.46 Purmorphamine turned out to target hedgehog (Hh) signaling via direct binding to the Hh receptor smoothened (agonism).47 However, therapeutic utility of this small molecule might be limited as it would require local drug delivery (the bone) to avoid systemic side effects. Several other small molecules have been identified thus far and are capable of stimulating MSC differentiation, including the PPARγ agonist rosiglitazone, the glucocorticoid dexamethasone, ascorbic acid, retinoic acid, vitamin D3, the cyclooxygenase inhibitor indomethacin, and many more.6 Cardiac Progenitor Cells. For the heart, the outlook for a regenerative therapy was bleak for many years, until the discovery of endogenous SCs in 2003 and more recently the demonstration of direct reprogramming in the heart in vivo as well as the discovery that adult terminally differentiated cardiomyocytes could be pushed to proliferate again.48 Since their initial discovery, several laboratories have now reported the identification of stem cell-like cardiac progenitor cells (CPCs). Methods of isolation from adult heart include using markers such as c-Kit or Sca149−51 or phenotypic approaches such as cardiosphere formation52,53 or isolation of a side population through efflux of the Hoechst 33342 dye.54−56 There is no absolute consensus in the field, however, of what markers define these CPCs, nor is it understood how these cells can be proliferated or differentiated in vitro or in vivo and what cardiovascular lineages they may develop into. Nonetheless, transcriptional profiling of CPCs isolated by different means did show overlap in marker expression, indicating that CPCs express a subset of cardiac specific transcription factors such as Isl1, Gata4, Nkx2.5, or Mef2c.49−51 Moreover, a recent report has suggested that c-Kit+ cells represent an earlier developmental stage of the other CPC classes described.57 Additionally, the epicardium has been suggested to be a source of progenitors that can be mobilized by thymosin β4.58 Functionally, these various CPCs are mobilized after infarct in the mouse and may contribute to new myocardium, yet their response is too inefficient to overcome the loss of cardiomyocytes or to restore cardiac function.49,59 A recent report suggested that c-Kit+ cells are essential for heart regeneration in certain models of cardiac injury, indicating that these cells indeed hold promise as a therapeutic target.60
Given the relatively recent discovery of these CPCs, little is known about how these cells may be expanded or mobilized in vivo to drive a regenerative response. Since CPCs are available in limited quantities, several high-throughput assays have been developed based on embryonic stem cell models to identify molecules that may drive cardiac differentiation at ESC stages that resemble the adult phenotype. One series of small molecules, the 1,2-isoxazoles (i.e., Isx-1, Figure 3), has been extensively characterized for tissue repair in the heart after infarction. Isx-1 was initially identified in a large-throughput screen using Nkx2.5 activity as a phenotypic readout for cardiac fate.61,62 When injected in vivo they activate a cardiac program in Notch-activated epicardium-derived progenitor cells (NECs). Unfortunately, Isx-1 seemed to induce a cardiac program in progenitors under homeostasis conditions but was not able to induce a regenerative response through NECs after injury. Isx-1, however, still had a beneficial effect on heart function after infarct, and subsequent work demonstrated that Isx-1 rather had a protective role on cardiomyocytes during ischemia by targeting GPR68, a pH-sensing GPCR.63 The combined work by Schneider and colleagues is one of the few examples in the heart where an in vitro screen has yielded a novel target and a small molecule that has benefits in vivo. Nevertheless, cardiac regeneration screens in zebrafish and ESC systems have yielded other cardiogenic small molecules including BMP, TGFβ, and Wnt inhibitors.64−68 Aside from Isx-1, the class of Wnt/β-catenin inhibitors is most likely the best candidate for an in vivo regenerative approach given the fact that Wnt signaling has been shown to maintain embryonic cardiac progenitors in a proliferative state.69 Inhibition of Wnt would thus drive differentiation of progenitors to a cardiomyocyte phenotype, and this is what is observed in the small molecule screens. In fact, two small molecule inhibitors of the Wnt pathway have been studied as drug candidates to reduce adverse effects of myocardial infarction, namely, pyrvinium and ICG-001.70,71 Similarly to Isx-1, these molecules did not seem to have an effect on scar size of the infarcted heart, yet they mediated improved cardiac function after infarction. The underlying mechanisms appeared to be different for both molecules. Pyrvinium activated a cardiomyocyte proliferation response, whereas ICG-001 targeted epicardial cells that contribute to improved function. While both studies demonstrate proof-of-principle for Wnt inhibitors for cardiac repair after injury, neither documented a clear underlying mechanism, and further work is warranted to understand whether there is a true regenerative response. Moreover, these studies should be repeated with different, more drug-like Wnt inhibitors that are now widely available,72−74 since at least pyrvinium is showing high toxicity in animal studies. 71 Together, these efforts demonstrate that in vitro screens for cardiac repair indeed represent useful alternatives to identify novel targets and molecules for regenerative medicine in the heart. Neural Stem Cells. An exciting finding in the neurobiology field has been the fact that neuronal cells and tissues are capable of regeneration. Mainly two regions in the adult mammalian brain are known to contribute to neurogenesis, i.e., the subventricular zone (SVZ) that surrounds the lateral ventricle and the subgranular zone (SGZ) within the hippocampal dentate gyrus. In fact, adult neural stem cells (NSCs) in these regions of the brain provide a pharmacological handle for noninvasive neuronal regeneration.19,75 Strikingly, many lines of evidence underline a causative link of adult hippocampus 61
dx.doi.org/10.1021/cb4008277 | ACS Chem. Biol. 2014, 9, 57−71
ACS Chemical Biology
Reviews
over, such antidepressants were reported to increase the number of neural progenitor cells.86 Another example of a marketed drug with neurogenic activity is the oral type II antidiabetic biguanide metformin. Metformin activates the atypical protein kinase C (aPKC)/CREB-binding protein (CBP) pathway in the liver via AMPK.87 After CBP was identified as an important player in neuronal precursor development, which required the activation by aPKC, Miller et al. reasoned that metformin could promote neurogenesis.88 Indeed, metformin enhanced neurogenesis in a CBP-dependent manner in adult mice and improved spatial memory function linking neurogenesis to the hippocampal region. An extraordinary in vivo approach was realized in a quest to identify small molecules that promote neurogenesis in the SGZ (mice) by screening ca. 1000 compounds.89 Compound P7C3 (Figure 3) was among the most effective candidates and turned out to improve the survival of newly formed neurons via protection from apoptosis, but it did not affect NSC differentiation. Although the molecular target needs to be identified, Pieper and co-workers managed to further optimize this molecule by medicinal chemistry efforts.90 Optimization was primarily targeted at improved potency, reduced toxicity, and stability. In fact, optimized compounds were orally available, nontoxic, stable, and capable of penetrating the blood brain barrier, an essential feature for the treatment of CNS disorders. Pieper’s approach underlines the beauty of in vivo screen for regenerative agents, accepting obvious drawbacks of low throughput and the inability to separate binding to therapeutically relevant target (pharmacodynamics) from pharmacokinetic effects. In summary, for several adult stem and progenitor cells, a large body of fundamental research has already led to the discovery and development of various small molecules that may serve as potential regenerative drugs. Most of these examples represent modulators of hematopoietic SCs with compounds affecting their fate (e.g., TPO mimetics), number, or quality. Nevertheless, several promising concepts and small molecules have been discovered for organs with less inherent regenerative capacity, either from medium-throughput phenotypic screens in stem cell models (e.g., Isx-1), or animals (e.g., P7C3) or by knowledge-based approaches (e.g., metformin).
neurogenesis and psychiatric and neurologic disorders, including depression and schizophrenia. Therefore, unraveling neurogenesis on the molecular and cellular level will provide attractive entries into therapeutic strategies for the treatment of cognitive and neurodegenerative diseases.76 Three main approaches have been pursued in the past for identifying mechanisms, molecular targets, and small molecules for neurogenesis: (1) targeting NSC self-renewal and proliferation, (2) NSC protection, and (3) stimulation of NSC differentiation toward specialized neuronal and glia cells. Many molecular mechanisms and small molecules have been identified that modulate in vitro NSC self-renewal, proliferation, and differentiation (comprehensively reviewed elsewhere).6 In order to identify novel compounds that specifically affect endogenous NSCs, Ding and Schultz performed an imagebased screen in multipotent hippocampal neural progenitor cells and identified neuropathiazol as a potent stimulator of neuronal differentiation (Figure 3).77 Further optimization of this compound resulted in KHS101, which promoted neuronal differentiation in vivo (rats) and appeared to function through targeting the TACC3-ARNT2 axis.78 A different study sought to target late-stage multiple sclerosis (MS) by screening for molecules that differentiate oligodendrocyte precursor cells (OPCs) to stimulate myelination, which does not occur in MS.79−81 To identify small molecule inducers of OPCs, an image-based screen in primary optic nerve-derived OPCs (rat) was done and furnished the anticholinergic drug benztropine as a remyelination enhancer (Figure 3). Benztropine significantly decreased disease severity in clinically relevant models of MS, and its mechanism appeared to depend on anti-muscarinergic activity. The discovery of benztropine is an impressive example of how cell-based high-throughput approaches can translate into in vivo-relevant regenerative agents through targeting adult progenitor cell populations. Aside from differentiation of NSCs to specialized neuronal cell types, neuritogenesis is part of the differentiation process (i.e., maturation), which leads to branching of neurites. Such neurotrophic processes are important for the restoration of neuronal viability and neuronal networks.82 Recently, Dakas and co-workers designed and evaluated focused compound libraries of iridoide glycosides and sesquiterpenoids in cultures of primary hippocampal neurons and ESC-derived motor neurons. This work yielded promising compounds that induced neuronal complexity, but further work is required to elucidate mechanisms of action.83 However, the lack of a complete understanding of how to chemically control NSC fate in a selective fashion represents a major hurdle, and the above-described neuronal differentiation agents need to be evaluated for safety and efficacy in humans. Therefore, such compounds are currently valuable chemical biology tools to decipher differentiation processes, but their therapeutic utility is unclear. This situation is different with antidepressants as they are clinically used drugs and have proven safety and efficacy. Interestingly, the first observations of neurogenic capacities in the adult mammalian brain were made in the context of depression and antidepressant therapy, respectively. Antidepressants such as the selective serotonin (fluoxetine) and noradrenaline (reboxetine) reuptake inhibitors and the monoamine oxidase inhibitor tranylcypromine have been shown to increase neurogenesis in the rat hippocampus (Figure 3).84 For fluoxetine, it has subsequently been proposed that this neurogenic activity could be linked to behavioral effects of chronic antidepressant therapy.85 More-
■
MOBILIZATION, HOMING, AND ENGRAFTMENT OF STEM CELLS Targeting adult stem cells for endogenous repair has attracted much attention in the past decade, mainly due to the appreciation of a link between adult SC behavior and an animal’s physiology as outlined by Miller and Kaplan.91 A large body of work documents the concept of SC recruitment and their participation in tissue regeneration via endogenous growth factors, hormones, and other physiological cues. In fact, based on these principles, successful translation to the clinic has already been achieved. Examples come from the hematopoietic system in the context of treating certain forms of anemia (e.g., G-CSF). In general, promoters of SC mobilization can potentially be useful to harvest large amounts of SCs for transplantation as well as to boost regeneration at remote sites of injury. For successful regeneration, mobilized (or transplanted) adult SCs subsequently must find (i.e., homing) and then functionally integrate (i.e., engraftment) into the damaged tissues, processes that are for instance mediated by cell surface proteins that support recognition of SCs or injured tissue, respectively.9 However, as with regenerative approaches in 62
dx.doi.org/10.1021/cb4008277 | ACS Chem. Biol. 2014, 9, 57−71
ACS Chemical Biology
Reviews
Figure 4. Small molecules for mobilization, self-renewal, homing, and engraftment of hematopoietic stem cells (HSCs). Mobilization of HSCs: Granulocyte-colony stimulating factor (G-CSF) mimetic (SB-247464) and C-X-C chemokine receptor type 4 (CXCR4) antagonism (plerixafor). Homing and engraftment: enhancing stromal cell-derived factor-1 (SDF-1) effects through inhibition of degradation by dipeptidyl peptidase 4 (DPP4) (diprotin A, sitagliptine). Self-renewal and engraftment: DPP4 inhibition (diprotin A) and stimulation of prostaglandin signaling (dmPGE2, EP2 = PGE2 receptor). Green arrow = activation. Red bar = inhibition.
(2008) as a combination therapy with G-CSF to increase HSC yields for autologous transplantation, although it was initially developed as an anti-HIV drug candidate.96−98 Due to plerixafor’s unfavorable pharmacokinetic profile, efforts were put into replacing the large cyclams that are positively charged under physiological conditions. Many second generation, orally available CXCR4 antagonists were developed to date, with few already in clinical trials.99,100 In a similar fashion, although functionally in contradiction to the above-described, mobilization of endogenous HSCs has been described by enhancing the CXCR4/SDF-1 interaction. Membrane-bound dipeptidyl peptidase 4 (DPP-4) hydrolyzes SDF-1, and therefore, pharmacological inhibition of this enzyme by diprotin A increases local SDF-1 levels and ultimately improves homing and engraftment of HSCs in mice (Figure 4).101 The FDA-approved type II antidiabetic sitagliptine also boosts activity of hematopoietic cytokines, which showed benefit in recovery of HSCs after chemotherapy (mice).102 Aside from CXCR4/SDF-1 as a central pathway for mobilization, homing, and engraftment of hematopoietic stem and progenitor cells (HSPCs), the VLA-4 (very late antigen 4, α4β1 integrin)/VCAM-1 (ligand) axis is a closely related pathway, which plays a similar role. First evidence for an effect on HSPC mobilization through inhibition of VCAM-1 binding to VLA-4 has been provided using an anti-VLA4 antibody.103 Ramirez and co-workers subsequently demonstrated that the small molecule VLA-4 inhibitor BIO5192 increases mobilization of murine HSPCs. Moreover they showed that this effect is synergistic with G-CSF and plerixafor treatment.104 Finally, North and co-workers made a significant contribution to the field by identifying prostaglandin signaling as a regulator of HSC fate. From a phenotypic screen (ca. 2,500 compounds) in zebrafish they discovered that prostaglandin E2 increases HSC numbers in vivo.105 Notably, just like all
general, whether recruitment of endogenous stem cells for tissue regeneration is a viable (and druggable) strategy decisively depends on the respective organ’s natural regenerative capacity and mechanisms involved. Hematopoietic System. As described above, the clinical use of HSC-based therapies requires culturing techniques and protocols that enhance proliferation and survival of HSCs and, ideally, would also rely on improved processes of homing and engraftment after transplantation. Granulocyte-colony stimulating factor (G-CSF) has been used clinically for many years to treat neutropenia as a consequence from cancer chemotherapy or chronic diseases and acts through stimulation of granulocyte production from the bone marrow. It is moreover used to harvest large numbers of HSCs for allogeneic or autologous transplantation.92−94 Additionally, G-CSF-induced mobilization of HSCs holds promise for regeneration of nonhematopoietic tissues such as the myocardium, brain, or other cells that have been lost from ischemia. The idea is that mobilized HSCs home to the site of injury, where they then engraft and differentiate to regenerate the respective tissue. Alternatively, they might provide paracrine signals that drive tissue regeneration. Different forms of recombinant human G-CSF are already available (e.g., filgrastim, lenograstim), but their short plasma half-life and chronic effects on the hematopoietic system still represent major limitations. Small molecule alternatives would be highly advantageous, and the discovery of SB-247464 (Figure 4) as a growth factor mimic has demonstrated general feasibility of such a strategy.95 G-CSF, at least in part, exerts its mobilizing effect through interference of C-X-C chemokine receptor type 4 (CXCR4) with its ligand stromal cell-derived factor-1 (SDF-1). Notably, this effect is further boosted by synergistic action via treatment with CXCR4 antagonists such as the macrocyclic bicyclam AMD3100 (plerixafor). In fact, plerixafor is FDA-approved 63
dx.doi.org/10.1021/cb4008277 | ACS Chem. Biol. 2014, 9, 57−71
ACS Chemical Biology
Reviews
Figure 5. Selected small molecule stimulators of cardiomyocyte and β-cell proliferation.
prostanoids, PGE2 is heavily metabolized, thus making in vivo applications difficult. 16,16-Dimethylation protects from oxidative metabolism, and thus, dmPGE2 is not a good substrate for the major (inactivating) metabolism through the action of prostaglandin dehydrogenase (Figure 4). This synthetic PGE2 derivative exhibits a prolonged half-life and was used for ex vivo treatment of HSCs, which more efficiently engrafted after transplantation.106,107 Heart. It is tempting to speculate that small molecules can be developed to mobilize SCs and to improve their integration in the infarct site of the heart. Very little is known about this mechanism for the heart and thus no drug-like molecules are available, but quite a few lessons can be learned from cell transplantation methods into the heart. Numerous efforts toward cell-based therapies have been taken to repair the damaged tissue by injection of a variety of SC types, ranging from bone marrow-derived stem cells, MSCs, and even ex vivo expanded cardiac SCs.108 While most of these approaches have a beneficial effect on cardiac function after myocardial infarction, the improvement observed is quite modest. The cells rarely contribute to new myocardium even though they are present in the heart after their delivery, thus questioning the actual regenerative benefit. Several reasons for the lack of tissue repair have been postulated, from poor survival, lack of integration, to failure to differentiate to myocytes in vivo. A different hypothesis was recently discussed by Zhau and colleagues: It does not matter what stem cell is injected, there is always some beneficial effect, suggesting that maybe not the cells themselves but rather their secretome helps in SC homing, differentiation, or even cardiomyocyte protection in the ischemic heart.108 Indeed, MSCs for example have been found to increase conduction via a paracrine mechanism and similarly Sca1+ SCs drive cardioprotection in a paracrine fashion.109,110 Since these different SC types have the ability to improve cardiac function, their secreted factors present interesting targets for drug therapy. To date, little is known about what kind of factors would mediate cardiac repair or how they would drive repair, yet elegant studies proposed by Karp and
colleagues should be able to identify key secretome factors involved.44 Aside from such large-scale experiments, a few candidates of these factors can be identified from the literature. SDF-1 for example drives improved conduction after infarct and may do more than just improving conduction, as SDF-1 is also of importance for homing of other progenitor cells, such as for example endothelial progenitors that express CXCR4 and home to injured heart.111 Other potential factors that not necessarily drive homing of SCs include VEGF and GDF11, which mediate cardiomyocyte size and neovascularization, respectively.112,113 As the secretome of these injected cells is further explored, more targets will be identified, and some of these could regulate homing or mobilization of endogenous stem cells to drive repair.
■
TARGETING STEM CELL NICHES Mounting evidence supports the role of extrinsic cues from an adult stem cells’ environment to critically influence its function in tissue maintenance and regeneration. This microenvironment is commonly called ‘stem cell niche’ and provides another potential handle to therapeutically control regenerative processes. The niche is a complex and dynamic system that provides spatial and temporal cues controlling the fate of tissue stem and progenitor cells regarding their quiescence or proliferation, self-renewal or differentiation, retention or migration, and cell death or survival, as comprehensively reviewed by Wagers.40 The composition of a given niche typically comprises the extracellular matrix (ECM), non-stem/ progenitor cells, such as stromal support cells and, of course, many cytokines, hormones, and growth factors. Moreover, physical factors such as temperature or oxygen tension contribute to a specific niche environment. Notably, the constitution of the niche eventually depends on the respective tissue as well as the current (patho)physiological state.40 Here, we defined small molecules that affect the expansion, mobilization, homing, or engraftment of adult stem and progenitor cells (see above) within the niche through direct targeting of adult stem cells, yet not as bona fide ‘niche modulators’. Nevertheless, such compounds provide insights 64
dx.doi.org/10.1021/cb4008277 | ACS Chem. Biol. 2014, 9, 57−71
ACS Chemical Biology
Reviews
genes p15, p16, and p21, indicative of a direct regulation of proliferation.121 The combination of cardiomyocyte proliferation and regeneration models in fish, mice, and pluripotent stem cells provide a powerful collection of biological assays to rapidly discover novel targets and small molecules that drive proliferation of cardiomyocytes. Pancreatic β-Cells. Despite their small numbers in the pancreas, β-cells are key players in the metabolic regulation of glucose homeostasis through the secretion of insulin. Their loss or dysfunction is central to the etiology of diabetes. Although multiple pharmacological therapies for type 1 and 2 diabetes exist, none are able to prevent the progressive decline in β-cell function, thus the long-term complications associated with this disease such as major damage to kidneys, eye, vascular, and nervous systems. Restoring lost or dysfunctional insulinproducing β-cells would therefore provide a true causative solution.23,122 Although human islet transplantation has been successful in clinical practice, there are many shortcomings associated such as the need for immunosuppression, scarcity of donors, and graft failure. Stem cell-based technologies for transplantation of β-cells or progenitor cells might overcome some of these limitations, also with the help of small molecules.23,123,124 In situ pancreas regeneration by proliferation of existing βcells has been verified, and lineage tracing studies have shown that new β-cells originate from the proliferation of β-cells as opposed to differentiation from endogenous stem cells (i.e., neogenesis).125,126 To date, several signaling pathways, growth factors, hormones, and small molecules have been reported to control β-cell proliferation.127 For instance, the glucagon-like peptide-1 (GLP-1) analogue exenatide (incretin mimetic) stimulates both β-cell proliferation and neogenesis, resulting in increased β-cell mass in diabetic rats.128 Similarly, blocking GLP-1 degradation by small molecule inhibitors of DPP-4 (e.g., sitagliptin, Figure 5) improved β-cell function in diabetic patients.129 Long-term studies will have to determine the benefits of targeting the incretin axis for the treatment of type 2 diabetes. Another druggable target in the context of replenishing β-cell mass emerged from a recent finding that the protease BACE2 is involved in the control of functional β-cell expansion in mice.130 The 1,3-thiazine CmpdJ (Figure 5) was identified as a highly potent BACE2 inhibitor with good selectivity over other proteases such as cathepsin D, but with rather poor selectivity over BACE1, which in contrast to BACE2 is highly expressed in the brain and fulfills a unique role in the generation of polymerized amyloid β-peptides.131 CmpdJ served as an excellent tool compound for the validation of BACE2 as a functional player in β-cell expansion. Extensive data from BACE1 inhibitor design as a prominent Alzheimer’s disease drug target and the availability of crystal structures of both BACE1 and BACE2 will certainly expedite the rational design of promising leads for sustaining studies in human.131 Among the very first screening-based attempts to identify small molecule stimulators of β-cell proliferation was a highthroughput screen (ca. 850,000 compounds) in growtharrested, reversibly immortalized mouse β-cells.132 These cells were transformed with SV40 TAg that could be induced by tetracyclin, which led to the proliferation of β-cells, but underwent growth-arrest upon tetracyclin removal. Although this system did not truly recapitulate in vivo quiescent β-cells, it still furnished several small molecules that induced re-entry into
into physiological, niche-relevant regulatory mechanisms as exemplified with G-CSF, plerixafor, diprotin A, or dmPGE2. Well-studied niches such as from the bone-marrow (HSCs), intestinal crypt or skeletal muscle as well as the hair follicle bulge may provide a fruitful ground for learning and extrapolation to other organs. Therefore, targeting the niche microenvironment provides many options yet requires further investigation for rational development of small molecule ‘niche modulators’ for a given organ of interest, such as the heart or pancreas.
■
SOMATIC CELL PROLIFERATION Regeneration not only may be achieved through targeting multipotent SC populations but also can be attained by stimulating cell division of somatic cells within the tissues. Although for many organs it has been long believed that cells cannot re-enter the cell cycle, recent evidence confutes this concept, suggesting a higher degree of cellular plasticity than originally thought. Therefore, boosting proliferation as a means of tissue regeneration is deemed a straightforward in vivo therapeutic approach and has been actively pursued, already yielding results with promising small molecules. Pro-proliferative agents are of particular interest for organs of very limited proliferative activity (e.g., heart), whereas for example several tissues of the gut (e.g., intestines or liver) already exhibit pronounced regenerative capacities via proliferation. Obviously, such therapeutic approaches should be carefully evaluated for enhanced risk of uncontrolled cell growth, potentially bearing a risk for tumor formation. Mitogenic activity not selective to desired cell or tissue types certainly raises questions of in vivo safety. Cardiomyocytes. In the heart, the turnover of adult myocytes in human was first described in 2009, and several studies subsequently showed that mouse myocytes also divide during adulthood, albeit on a rather limited basis under homeostasis conditions.48,114,115 Interestingly, the zebrafish heart regenerates spontaneously after injury, which was recently demonstrated to be the case in neonatal mice hearts as well. This capacity is however lost during adulthood.116 The zebrafish presents a good model for chemical screening and provides years of knowledge on the biology underlying heart regeneration. In the fish, insulin-like growth factor (NBI31772) and hedgehog agonists (smoothened agonist, SAG) were identified from an in vivo screen, in search for molecules that drive cardiomyocyte proliferation (Figure 5).117 Another screening approach taken in an ESC-derived system tried to address some of these questions in the mammalian system and yielded four classes of compounds, including p38 MAPK (SB203580), GSK-3β (CHIR99021), ERK, and CamKII inhibitors that were driving proliferation. Translation of fish and in vitro ESC data to a mammalian in vivo model of infarction remains crucial, but it is encouraging to see that cardiomyocyte-specific GSK-3β deletion in the heart in vivo leads to hyperproliferation of adult cardiomyocytes.118,119 The fact that heart regeneration and cardiomyocyte proliferation now can also be studied in the mouse in vivo has triggered some interesting work, driven by the search for factors that control this early regenerative potential and how that could translate to adult life. One such biological target is the Hippo/Yap pathway, which is required for neonatal regeneration to occur and provides a tractable target for small molecules.120 Another target at the helm of heart regeneration is Meis1, a transcription factor that directly regulates the Cdk 65
dx.doi.org/10.1021/cb4008277 | ACS Chem. Biol. 2014, 9, 57−71
ACS Chemical Biology
Reviews
cell cycle and proliferation. For instance, phorbol esters as protein kinase C activators and a class of thiophene pyrimidines as Wnt signaling agonists could induce proliferation. Moreover, the L-type calcium channel agonist BAY K8644 was identified as a β-cell proproliferative agent. However, these compounds inherit rather unspecific mechanisms of action and were only moderately active in primary islet cells. A much more promising compound, diarylamide WS6, was later disclosed by the same group and appeared to be less promiscuous in action (Figure 5).133 Importantly, WS6 also enhanced proliferation in primary rodent and human islets and ameliorated type 1 diabetes in vivo (mouse model). Affinity-based target identification led to Erbbinding protein 1 (EBP1) and IκB kinase ε (IKKε) as putative effectors of WS6’s mitogenic activity in β-cells. An exciting small molecule approach to increasing β-cell mass in vivo has been the discovery of adenosine kinase (ADK) inhibitors 5-iodotubercidin and ABT-702 from a small-scale, focused cell-based screen (850 compounds) (Figure 5).134 Annes and co-workers used primary rat islet cells and quantified PDX1 (β-cell specific transcription factor) and ki-67 (proliferation marker) expression with the aim to selectively measure β-cell proliferation to resemble the in vivo situation. ADK inhibitors promoted β-cell proliferation in mouse, rat, and pig, and this activity was selective to β-cells but did not affect αcells, pancreatic polypeptide cells, or fibroblasts. In addition, the authors demonstrated β-cell proliferation by ADK inhibitors in vivo but did not observe effects on exocrine cell or hepatocyte proliferation. The authors hypothesize that ADK inhibitors activate mTOR pathways in β-cells in a unique fashion. While several promising concepts and the accompanying small molecules were reported to boost regeneration, human βcells exhibit limited proliferative responses to stimuli that dramatically increase β-cell mass in rodents. This should be considered when extrapolating data from rodents to humans.135 It remains to be addressed whether small molecules such as BACE2, ADK inhibitors, or WS6 can promote replication of healthy and diabetic β-cells in humans.
the identification of many valuable compounds such as dmPGE2 (zebrafish) or P7C3 (mouse). Although facing the inevitable downsides of low-throughput and the difficult elucidation of underlying mechanisms and targets, such screens deliver bona f ide regenerative compounds capable of inducing the desired phenotype in vivo. Moreover, lessons learned from the clinic led to the discovery of novel regenerative drugs along with critical mechanisms of action involved, as illustrated with the neurogenic activity of several distinct classes of antidepressants. Such findings not only stimulate rational starting points for designing novel regenerative molecules but also imply repurposing strategies of approved drugs. One exciting prospect in the regenerative medicine field comes from direct reprogramming, where one somatic cell can be directly converted to another. To date, neurons, β-cells, and cardiomyocytes have been generated from fibroblasts through the introduction of a selected set of fate-specific transcription factors, similar to the Yamanaka approach to establish pluripotency from somatic cells. 136−138 The fact that reprogramming works in vivo, at least for the pancreas and the heart, is transformative for the entire field and opens up a whole new series of biological targets for drug development. Together, the development of regenerative drug candidates requires a solid understanding of the underlying pharmacologically relevant signaling pathways and/or cellular targets. Many of the herein presented ‘success stories’ corroborate this as they are already translated to in vivo regeneration. A key future challenge of small molecule-based in vivo regenerative approaches that certainly needs to be addressed includes the translation to humans. Moreover, safety issues owing to the agents’ cell or tissue specificity, selectivity, and their individual mechanisms of action that typically modulate cell fate decisions need to be assessed.
CONCLUSIONS AND PERSPECTIVE While significant progress has been made using small molecules for in vitro stem cell culture with the aim of therapeutic transplantation, replenishing lost and/or dysfunctional tissue in vivo is still in its infancy. This is mainly due to a limited understanding of mechanisms that control tissue homeostasis, repair, or regeneration. However, building on current knowledge in stem cell biology and regenerative medicine, a plethora of different approaches has already resulted in the discovery and development of in vivo active small molecules. Because of the extensive research on the hematopoietic system for decades, knowledge-driven drug discovery campaigns were realized and furnished marketed small molecules such as the TPO mimetic eltrombopag or the CXCR4 antagonist plerixafor. Especially, HSCs can potentially serve as a model for organ regeneration and lessons learned may possibly be extrapolated to other organs. Nevertheless, even with less information in hand, modern phenotypic screening assays that recapitulate distinct processes of tissue regeneration have proven successful. For instance, by chemical screening in primary adult SCs or pluripotent SCderived progenitor cells small molecules such as stemregenin1, kartogenin, or benztropin were discovered, all of which subsequently revealed efficacy in vivo. Aside from cell-based screening approaches, in vivo models of regeneration allowed
Notes
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected].
■
The authors declare no competing financial interest.
■ ■
ACKNOWLEDGMENTS Financial support by the Bundesministerium für Bildung und Forschung (BMBF) is greatfully acknowledged.
66
KEYWORDS adult stem cells: tissue-specific stem or progenitor cells that can differentiate into several cell types within their lineage (i.e., they are multipotent) and play central roles in tissue homeostasis and regeneration upon injury. They exhibit pronounced differences in their regenerative potentials depending on the organ, with hematopoietic and mesenchymal stem cells as highly regenerative systems, whereas most other organs are endowed with limited regenerative capacities. endogenous regeneration: comprises physiological (i.e., in vivo) processes of cell, tissue, or organ regeneration upon injury as opposed to ‘ex vivo regeneration’ that involves transplantation techniques mobilization, homing, and engraftment: a process of stem cell activation, possible expansion, and release from their niche environment (mobilization) to participate in regenerative processes. Such mobilized stem cells must subsequently dx.doi.org/10.1021/cb4008277 | ACS Chem. Biol. 2014, 9, 57−71
ACS Chemical Biology
Reviews
find their target site(s) either within the same tissue or at remote sites of injury in the body (homing) and ultimately be functionally integrated (engraftment) in that respective tissue, which typically includes distinct steps of differentiation and maturation. proliferation: a fundamental process of cell growth and development where one somatic cell divides into two daughter cells. In the context of regeneration, proliferation represents a powerful means to replenish lost or damaged cells and tissues. regenerative medicine: deals with the replacement or regeneration of human cells, tissue, or organs to restore and establish normal function reprogramming: as referred to in this review, the direct transformation of one specialized, terminally differentiated somatic cell type to another one without going through a stem cell-like status. Reprogramming, however, is also wellknown in the context of generating induced pluripotent stem cells from terminally differentiated cells of various types using combinations of distinct reprogramming factors. small molecules: (physico)chemically well-defined organic substance with a molecular weight of
