Stem cell biology and drug discovery

There are many reasons to be interested in stem cells, one of the most prominent being their potential use in finding better drugs to treat human disease. This article focuses on how this may be implemented. Recent advances in the production of reprogrammed adult cells and their regulated differentiation to disease-relevant cells are presented, and diseases that have been modeled using these methods are discussed. Remaining difficulties are highlighted, as are new therapeutic insights that have emerged.

differentiated cells that can be used as models on which to screen new drugs. One motivation for this is the wide spread recognition that the drug discovery process as practiced in most pharmaceutical companies is ineffi cient, at best, and, in the past decade or so, has struggled to meet the need for new drugs. In addition, there have been a number of famous cases in which already marketed drugs have been found to have unanticipated side effects. Standard preclinical drug safety testing relies exclusively on administering drugs to two nonhuman animal species, and it is possible that safety studies on validated human cells might help avoid unexpected drug toxicities.

Three key advances
From our perspective, the interest in stem cell biology as a route to novel therapeutic drugs arose from the convergence of three separate lines of investigation. First there is evidence that pathways that regulate embryonic development and, hence, act in large part on tissue stem and progenitor cells are also disrupted in adult disease [6,7]. For example, the hedgehog signaling pathway, of vital importance in nervous system development, is hyper activated either by mutation or by ligand over expression in a significant percentage of human cancers [8]. More than 10 years ago, we showed that it was possible to identify druglike small molecules that inhibit hedgehog signaling and are effective in various cancer models [9,10], bringing together the worlds of develop mental biology and conventional drug identification. In fact, as recently presented at the American Association for Cancer Research meeting by Dr Ervin H Epstein, a derivative of the first hedgehog antagonist developed, vismodegib, has been shown to have positive results in a phase II clinical study for metastatic basal cell carcinoma. Other hedgehog antagonists have already entered the clinic, including several developed by major pharma ceutical companies [11]. The observation that there is a link between stem cells, their regulatory pathways, and disease has clearly piqued the interest of the pharma ceutical industry, and there is serious interest in develop ing modulators of other pathways, such as Wnt and Notch, that are active in the embryo.
The second trend followed from a seminal discovery made by Jessell and coworkers [12] on the specification of motor neurons and other neurons in developing mouse spinal cord. They established a key role for sonic hedgehogregulated signaling, and went on to show that the differentiation of motor neurons could be recapitu lated in culture by adding retinoic acid to mouse ESCs to generate spinal cord progenitors and then an activator of the hedgehog pathway [12]. That was achieved with a small molecule that potently activates hedgehog signaling [13]. The lessons learned from this study were that: (a) it is possible, at least some of the time, to control differentiation of ESCs; (b) small molecules that regulate differentiation can be found; (c) by correctly controlling properties of stem and progenitor cells, it is possible to contemplate making large numbers of a defined type of cell. This work also opened up the possibility of making large numbers of differentiated cells from mice engi neered to express human disease genes.
The third major advance was the reprogramming of adult cells to induced pluripotent stem cells (iPSCs), described by Yamanaka and coworkers [14,15]. The discovery that differentiated cells for example, dermal fibroblasts -could be induced to revert to a pluripotent state made it possible to avoid both the political and the practical difficulty of using human ESCs, of which supplies are limited. iPSC technology offers the prospect of capturing cells derived from a large number of specific types of prediagnosed adult patients, potentially at any age, and a correspondingly large number of controls in a format that can support an industrial level of screening, efficacy, and safety studies.

Stem cells as a tool for drug research and development
Stem cell biology is a rapidly growing field, and many excellent reviews of some of the topics covered here are available. In this article, we focus on using stem cells for drug research and development.
The central concept is that stem cells can provide a new means of studying the pathological basis of disease, screening for drug leads, testing candidate drug efficacy and safety, and selecting patient populations for clinical testing. The plan would be to identify a disease of interest and obtain skin biopsies or other tissue samples from patients with that disease. For each patient, iPSCs would be generated, expanded and (re)differentiated to the type of cells most affected in the disease of interest -for example, motor neurons for amyotrophic lateral sclerosis (ALS) or spinal muscular atrophy (SMA) -and to those most commonly affected by drug side effects (cardiac myocytes and hepatocytes). Once appropriate studies on disease mechanisms had been completed, screens could be set up to discover drug leads capable of correcting the disease phenotype. These screens may be phenotypic; for example, for ALS, a motor neuron survival screen could be appropriate. Hit compounds would be pursued by medicinal chemists (in the case of small molecule thera peutics) in the traditional way. But efficacy would continue to be tested on human diseased cells, and safety would be assessed in a preliminary fashion using corres ponding cardiac and liver cells. Once potential lead com pounds were identified, they would be tested on a broad sampling of individual patientderived diseased cells, along with cardiac muscle and hepatocytes. This would aid in deciding whether certain compounds were more likely than others to be active across a large percentage of patients or, at a minimum, in preselecting the particular patients most likely to respond to a specific agent. The cost of drug discovery could be considerably reduced if a greater percentage of compounds entering the clinic were approved as drugs as a consequence of having better drug targets, better safety profiles, or a more considered choice of patient population.
How can we decide if this new approach can really evolve into an improved system of discovering and test ing new drugs? Ultimately, the answer can only be provided in the clinic, and that will take a long time. How ever, prior to that, we will need to establish tech niques to (a) produce patientderived cells that are capable of multilineage differentiation; (b) regulate their differ en tiation into diseaserelevant cell types; (c) use the differ en tiated cells to learn more about diseases of interest; (d) carry out primary screens and other types of efficacy testing on those cells; (e) assess a small number of the best compounds against a large sampling of patientspecific diseaserelevant cells. These steps are explored in greater detail below.

Producing cells with broad differentiation potential: iPSCs
The original methods of adult cell reprogramming were based on the use of viral vectors that drive the expression of the four transcription factors -Oct3/4, Sox2, cMyc and Klf4 -identified by Yamanaka and colleagues. However, at least one of these -cmyc -has oncogenic potential [14,15], and these methods are also subject to the risk of insertional mutagenesis. This has led to efforts to produce iPSCs without genome modification. Most recently, a great deal of interest has surrounded a new method of reprogramming that is based on the addition of synthetic mRNAs encoding the four Yamanaka trans cription factors [16].
At least some of the concerns associated with repro gram ming would be avoided if it were possible to re program with just small molecules or proteins, and chemical biologists have also studied the reprogramming process. A large number of cocktails have been derived, all of which use different mixtures of small molecules and transduced genes (reviewed in [17]). The small molecules identified have typically replaced one or more of the reprogramming factors or have improved the efficiency of the overall process. Many of the screens have provided some insight into the mechanism of reprogramming. One example of such a screen was based on a simple experiment designed to identify a small molecule capable of replacing the transcription factor Sox2 [18]. Mouse embryo fibroblasts were transduced with retroviruses coding for Klf4, Oct4 and cMyc, but not Sox2. Under those conditions, no true iPSC colonies formed. The cells were then treated with agents selected from an annotated compound library enriched in small molecules that modulate intracellular signaling. The most potent hit was an inhibitor of transforming growth factor (TGF)β signal ing. The surprise was in the way it acted: it increased expression of Nanog, another transcription factor with reprogramming activity. Furthermore, it affected not the starting cell population, but a population of partially reprogrammed intermediate cells that appeared 1 to 2 weeks after virus addition. This work, along with many other reports, demonstrates that reprogramming can be achieved in several, perhaps numerous, ways, with cells traversing different paths of dedifferentiation via many transient states of partial dedifferentiation. These repro gram ming intermediates are, in a sense, artificial, being created as a result of an artificial process. This concept will be explored later in the context of regulating a real biological process: cell differentiation.
Disappointingly, at the time of writing, it has not been possible to reprogram cells completely with chemicals, although small molecules that replace individual trans cription factors have been found. Is this because it is difficult to replace the activity of transcription factors effectively with small molecules? That may be true, although, as pointed out above, TGFβ inhibitors act by regulating the expression of a transcription factor. Is it because it is difficult to find combinations of small molecules that complement one another in the way that the transcription factors can? Is it just due to a lack of experimental insight into how best to replace these particular transcription factors? Answering these ques tions will be important because there are many other circumstances, reviewed later, in which small molecule modulation of cell fate could be valuable.

How useful are iPSCs?
In the past few months, a spate of publications have highlighted problems that might be inextricably linked to reprogramming itself, perhaps independent of the parti cu lar method used [1922] (reviewed in [23]). These include defects related to mutations, gene copy number variation, and incomplete resetting of DNA methylation. Some of these abnormalities may persist in differentiated cells produced from the iPSCs; some may be selected against by repetitive passaging. It is probably fair to say that iPSCs will be difficult to use therapeutically until these issues are resolved.
In spite of this significant concern, iPSCs may still have significant value in drug discovery. However, in that context, there is another potential problem. iPSC clones, even those prepared from a single patient, vary in their capacity to give rise to differentiated cells. Such variability has been seen previously with human ESC lines [24], which can show significant differences although they all meet the standard criteria for ESCs. That is, although they were all able to give rise to cells from the three germ layers in vitro and form teratomas in mice, some gave rise to endodermal lineages well, some gave rise to meso dermal lineages well, and so on. Thus, the standard criteria used to define pluripotency do not preclude line toline variability.
In an attempt to provide a systematic basis for charac terizing stem cell lines, Bock and colleagues [25] carried out an extensive bioinformatics comparison of 20 human iPSC and 12 human ESC lines, including DNA methyla tion patterns, microarray analyses, and a general differen tiation assay in which gene expression was analyzed in embryoid bodies derived from each line. On the basis of these data, it was possible to distinguish an average iPSC line from an average ESC line, although there was also considerable overlap. Importantly, the authors developed a scorecard based on a 500gene expression array to quantify the differentiation tendencies of each line. The scorecard predicted that two of the iPSC lines might have reduced ability to differentiate into neurons and this was confirmed experimentally in a study carried out by Boulting and colleagues [26], who, however, also showed that most iPSC lines, whether derived from healthy controls of different ages and sexes or from different types of ALS patients, could be induced to differentiate adequately into motor neurons. This suggests that the variability of the cell lines may not preclude their use in screening. What remains to be measured is the degree of variability in cell response to therapeutic candidates. Do motor neurons produced from several different iPSC lines, all from the same patient, have the same response to potential drugs? Are data collected from motor neurons derived from different individuals reliable enough to predict clinical responsiveness across patients? Information like this is essential for the approach being discussed here. It will also be essential to develop methods for reliably inducing the various types of differentiated cells from stem cells. In that aim, there is good alignment between scientists interested in drug discovery and those focused on regenerative medicine (cellbased therapy). Thus, there is a real need to understand how to produce cells that are sufficiently differentiated to (a) model pathological aspects of disease; (b) faithfully predict drug safety; and (c) integrate effectively into tissue when transplanted.

Embryonic development in a dish
Recent studies aimed at producing specific differentiated cells from ESCs or iPSCs have followed the principle established by Wichterle and colleagues [12] and attemp ted to recapitulate embryonic development in cell culture. At the core of this approach is the recognition that embryonic development occurs as a series of steps, with cells that have multipotential capacity becoming increas ingly differentiated ( Figure 1). However, even armed with this recognition, success has been somewhat mixed.
One instructive example is that of Kattman and colleagues [27], who published a very thorough paper describing a protocol to produce cardiac myocytes from ESCs and iPSCs in which they sequentially added morpho genic factors important in the appearance of cardiac muscle. They stressed a few general conclusions: (a) the first step of any differentiation procedure, the induction of the correct germ layer, must occur effi ci ently; (b) quantitative markers of different stages of develop ment are helpful; (c) the timing of activation or inhibition of various morphogenic pathways is critical, especially given that the very same pathway can have a stimulatory or an inhibitory influence at different times; and (d) the concentration of the inducing factors must be controlled carefully. In essence, this work confirms that the complex environment of the embryo can be reproduced to at least some degree. However, the authors also pointed out that there is significant variation among different cell lines so that protocols may have to be tailored to each, perhaps because individual lines may make variable amounts of their own inducing factors. This would be a significant hurdle if it were necessary to produce cardiac myocytes from tens or hundreds of patient lines for drug toxicity testing. Thus, finding a way of overriding this variability would be a valuable advance.
Again by adopting an analogous strategy, Studer and colleagues [28] have pursued methods for producing parti cular types of neurons efficiently. Importantly, they introduced a convenient way of regulating early neural induction by treating human ESCs, grown without standard feeder layers, with inhibitors of both TGFβ and bone morphogenetic protein (BMP) signaling [28]. This group went on to show the utility of this technique in the generation of dopaminergic neurons and motor neurons. Subsequent studies confirmed its utility in the derivation of cell types as diverse as neural crest [29] and floor plate [30]. The top schematic is generic and could be applied to any cell type. The lower paradigm is one that could be used to produce pancreatic β-cells and is taken from the work of Chen et al. [43]. DE, definitive endoderm; EP, endocrine progenitor; PP, pancreatic progenitor. Adding complexity to the culture environment Eschenhagen and Zimmermann [31] have pointed out that the field of tissue engineering first arose as a consequence of efforts to produce functional tissue for implantation. Over the past few years, many investigators have tried to apply the principles of tissue engineering to the problem of producing individual types of differ en tiated cells by making the cell culture environment more like in vivo conditions, essentially by making it more complex. VunjakNovakovic and Scadden [32] have sum marized the elements of a tissue engineering approach as including: (a) inducing factors; (b) extracellular matrix; (c) other cells, such as endothelial cells or stromal cells; and (d) physical factors, such as the rigidity of the tissue culture surface.
Various studies have incorporated some of these ele ments. As a simple start, numerous groups are interested in growing cells as threedimensional aggregates, as a kind of intermediate between standard culture conditions and the true in vivo setting. In essence, both embryoid bodies and neurospheres are based on this philosophy. Mei and colleagues [33] published a very extensive study in which ESCs were plated on a combinatorial set of substrates and adsorbed proteins. They discovered a few combinations that supported ESC growth and colony formation particularly well. Approaches like this will undoubtedly prove useful, including as a way of replacing feeder layers or for encouraging uniform growth and spreading of cells across the culture surface. Underhill and Bhatia [34] described attempts to microfabricate extra cellular matrix coated surfaces to allow cell growth, differentiation and survival. Several studies have empha sized the influence of the rigidity of the culture substrate. Gilbert and colleagues [35] found that muscle stem cells cultured on flexible hydrogel substrates like that found in real muscle retained more of their stem cell character istics and performed better in a muscle regeneration assay. In another interesting application of tissue engi neering principles, Domian and colleagues [36] induced differentiation of cardiac progenitors, purified them by fluorescenceactivated cell sorting (FACS), and plated them on a fabricated thin film, thereby constructing a contractile sheet of cardiac muscle. These methods may turn out to be valuable in producing cells that are mature enough to adequately represent cellular function or dysfunction, as will be highlighted below.

A transdifferentiation approach
A recent promising alternative way of producing differen tiated cells, from large numbers of patients if necessary, is by direct reprogramming -or transdifferentiationwhich is based on prior identification of transcription factors important in lineage specification (Figure 1). Just a few years ago, Zhou and colleagues [37] showed that pancreatic exocrine cells could be converted in vivo to pancreatic βcells by infecting them with adenovirus expressing three transcription factors, Ngn3, Pdx1 and Mafa, all known to be important for βcell development. Surprisingly, this occurred without proliferation of the exocrine cells, or even transient dedifferentiation to a progenitor cell state. Subsequently, Vierbuchen and colleagues [38] demonstrated that mouse fibroblasts, following treatment with lentivirus containing genes for three transcription factors expressed in the nervous system, Ascl1, Brn2, and Myt1l, could be induced to differ entiate directly to neurons. Neurons could also be derived from glial cells, which are embryologically more similar, by expression of only Neurog2, a transcription factor important in neural determination [39]. Ieda and colleagues [40] showed that expressing three transcrip tion factors important in heart development, Gata4, Mef2c, and Tbx5, could cause transdifferentiation of fibroblasts into cardiac myocytes.
These studies, and the profusion of those likely to follow in the near future, demonstrate fibroblasts need not be dedifferentiated completely to become other cell types. The potential advantage of this more direct approach, at least from a drug discovery perspective, is that more of the epigenetic modifications of patient derived cells might be preserved if it were possible to bypass complete reprogramming. Also, there is some hope that this method may make it easier to produce more mature cells than one that relies on reversion of cells to a more embryonic celllike state. Both of these differences could help in producing cells that more accurately model components of different diseases, although it is too early to judge how well the method will work. Can the fibroblasts be expanded sufficiently before viral transduction to allow for the generation of a sufficient number of differentiated cells? Can fibroblasts be obtained from older patients and still be trans differentiated?
A third possibility has arisen that is based on partial dedifferentiation with a subsequent differentiation step ( Figure 2). Reprogramming of mouse embryo fibroblasts is initiated but then aborted, and cells are put into a newly formulated medium that allows for the production of (in this case) cardiac myocytes [41]. Under certain circumstances, this method might allow for sufficient and rapid expansion of a type of progenitor cell still capable of multilineage differentiation.

Small molecule regulators of differentiation
A final way of inducing cell differentiation is, in a sense, less rigidly adherent to the notion of replicating the precise inducing conditions that underlie in vivo develop ment. The thinking behind this is that most investigators interested in therapeutics have the production of a single The question is whether this is true of differentiation as well, and if so, how could these pathways be identified? We and many others have adopted a screening approach in which stem or progenitor cells are treated with hundreds or thousands of small molecules and the effects on differentiation measured, generally by automated imaging. As reviewed previously [42], certain types of small molecule libraries are useful in these screens because, in principle, they allow for activation and in activation of many different intracellular signaling cascades. On the basis of this idea, Chen and colleagues [43] followed the general format of sequential differentiation outlined in Figure 1, but, rather than restricting them selves to a small set of morphogens, tested about 5,000 small molecules in a successful effort to find agents that increase the production of pancreatic progenitors from human endodermal cells. An extensive analysis of marker gene expression showed that the chemically induced progenitor cells were highly similar to the ones that appear in the embryo and were capable of progressing further through development, producing a small number of functional βcells.
The most effective small molecule hits in this screen were protein kinase C (PKC) activators. It will be interesting to determine if PKC activity is an essential part of early pancreatic differentiation in vivo. Alterna tively, PKC might be a crucial component of an alter native path from endoderm to pancreas that is not used in the embryo. Thus, manipulation of certain receptors or signaling pathways may allow cells to escape their rigid developmental boundaries, while rendering them still capable of reaching a normal developmental endpoint. If There are many paths from one differentiated cell to another. These include reprogramming to an iPSC followed by differentiation, transdifferentiation from one differentiated cell to another, and partial dedifferentiation to a cell that we call a PiPSC (partially induced pluripotent stem cell) followed by differentiation. this were true, it would have some farreaching impli cations. First of all, it raises the question of how to establish whether the cells that are produced by the different methods are actually the same or at least similar enough. Recalling the discussion of pluripotency of ESCs and iPSCs, by many criteria, a variety of cell lines were classified as being pluripotent, but the standard criteria must have been too loose since the lines are variable. The same could be true with differentiated cells produced by different methods, so it may be necessary to use an equivalent of the scorecard described by Bock and colleagues [25]. Or, since there are, for the most part, two uses for differentiated cells -modeling disease in vitro and functioning appropriately when transplanted -it should be possible to establish practical criteria.

Disease modeling using stem cells: can stem cells help us find better and safer drugs?
Assuming that issues with the production of pluripotent cells from patients' tissues, and the generation of differ entiated cells from them, can be resolved, what other con cerns remain? Unfortunately, there are many funda mental questions that have still not been addressed. One important issue relates to the degree of maturity of the cells that are produced. For instance, ALS and SMA are both motor neuron diseases, but they affect different motor neuron populations. In ALS, neurons innervating distal muscles are most sensitive while, in SMA, those innervating proximal muscles are most at risk. Further more, in both diseases certain rare populations of motor neurons are completely unaffected so, in theory, when trying to model those diseases, using very specific types of motor neurons would be most appropriate. In fact, motor neurons produced by the most common differ entiation protocols have a rather generic rostral cervical identity, although there is good reason to think that they can be induced to differentiate further by additional morpho gens [44]. Presumably, a transdiffer entiation approach in which the correct motor neuron pool specific transcription factors are expressed in the motor neurons could also be successful. This is not the only consideration, though. Typically, cells derived from pluripotent cells resemble their embryonic or immature counterparts (for example, [45]). Can they be induced to mature sufficiently to model adult disease? This has been hotly debated, especially in the context of late onset disease [46]. Many neurodegenera tive disorders, such as AD and PD, take decades to affect humans and even many months to affect transgenic mice, so is it reasonable to think that neurons derived from stem cells could be induced to adopt a disease phenotype? It is possible that even in the late onset diseases, some of the pathological changes, such as protein aggregation, occur long before clinical symptoms. Another possibility relates to the fact that many of these diseases are primarily sporadic and may be initiated by the presence of particular environmental factors. Exposing cells to high concentrations of, or prolonged incubation with, these factors might greatly accelerate the appearance of pathology in the cell culture environment. For example, the addition of cellular stressors, such as prooxidants or other compounds that compromise mitochondrial func tion, might bring on diseaserelated alterations [45,46].
Another important issue concerns the nature of the diseases that realistically can be modeled by applying a reprogramming or even a transdifferentiation method to patientderived cells. Naturally, monogenic diseases seem most amenable to this technique, and monogenic diseases that affect predominantly one cell type are likely to be better still. For these conditions, the expectation is that the reprogramming process will maintain the mutations involved, as will the differentiation protocol. However, what about diseases that are mostly sporadic and might involve epigenetic modifications of the genome? In those cases, reprogramming would tend to erase most of the epigenetic marks. Perhaps the transdifferentiation method will help in this regard, but this is not yet clear.
Certainly, the major degenerative diseases of the nervous system are primarily late onset and, while mostly sporadic in nature, are known to involve a small percentage of cases with well known diseasecausing mutations. One way forward that may be both doable and instructive is to establish an in vitro phenotype using the genetic variants of the disease first, and then test the sporadic cases to determine if there are culture condi tions that will produce the same disease pathology. Alterna tively, it might be possible to identify pathologyproducing cell culture manipulations that are informative about identifying the causative factors for the disease: for example, addition of certain insecticides may accelerate the onset of disease features in a PD model [45].
Starting just a few years ago, there have been many attempts to apply an overall stem cell strategy to the understanding of specific diseases. The typical starting point has been the production of patientspecific iPSCs. One of the first comprehensive reports was that of Park and colleagues [47], who derived them from patients with adenosine deaminase deficiencyrelated severe combined immunodeficiency, ShwachmanBodianDiamond syn drome, Gaucher disease type III, Duchenne and Becker muscular dystrophy, PD, Huntington's disease, juvenile onset diabetes, and Down syndrome/trisomy 21. More recent efforts have included production of iPSCs from patients with SMA [48], ALS [49], and Hutchinson Gilford Progeria Syndrome (premature aging, associated with vascular defects) [50,51]. A few illustrative cases will be presented. Rubin and Haston BMC Biology 2011, 9:42 http://www.biomedcentral.com/1741-7007/9/42

Nervous system disorders
One of the first examples in which an ESCbased approach (admittedly using mouse ESCs) contributed to a further understanding of disease mechanisms was that of ALS. Di Giorgio and colleagues [52] and Nagai and colleagues [53] established in vitro models of ALS by producing motor neurons from ESCs isolated from a transgenic mouse that carried a human superoxide dismutase mutation (G93A) found in a small percentage of patients with ALS. Although ALS is a late onset disease (decades in humans; approximately 4 months in mice), the authors, nonetheless, found a disease phenotypedeath of G93Aexpressing motor neurons was faster than that of wildtype motor neurons. In addition, they observed that astrocytes in the G93A motor neuron cultures appeared to secrete a toxic factor that further accelerated motor neuron death. The effect was selective in that interneurons were not killed by this factor. Subsequent work by Di Giorgio and colleagues [54] demon strated that mutant mouse astrocyteconditioned medium could also selectively kill human motor neurons produced from wildtype human ESCs. Thus, these investigators succeeded in modeling an adultonset neuro degenerative disease and in gaining some insight into molecular mechanisms that underlie the disease. What remains uncertain is whether this conclusion can be generalized to other genetic forms of human ALS and whether it is possible to establish an informative disease phenotype starting with sporadic cases of ALS.
Another interesting recent study was carried out by Marchetto and colleagues [55]. Many neurobiologists have been interested in using an iPSCbased approach to study autism spectrum disorders (ASDs), a group of related neurodevelopmental defects. However, while there are undoubtedly genetic factors underlying these diseases, they are complex, and environmental factors seem to play a major role. These investigators chose instead to investigate patients with Rett syndrome, which is asso ciated with impaired neural development about one year after birth and is caused by a mutation in the Xlinked gene MeCP-2. Children afflicted with Rett syndrome have some of the symptoms found in other ASDs, but it is frequently used for this type of study because it is a genetic, rather than sporadic, disorder. This clearly makes it amenable to an iPSC type of approach. The group produced iPSCs from patients and from them prepared a mixed population of neurons, including GABAergic inhibitory neurons and glutamatergic excitatory neurons. Reassuringly, they found that the reprogramming process erased the Xinactivation of the MeCP-2 gene, but it was reestablished during neuronal differentiation, just as was hoped. Next, they found that there was not a large defect in survival of the induced neurons, at least after 2 months in culture. Nonetheless, there was a significant decease in the number of glutamatergic synapses, recapitulating the failure to appropriately form or maintain a normal number of functional mature synapses seen in the syn drome. Finally, they showed that insulinlike growth factor 1 (IGF1), previously shown to have some ameliorative effects in a mouse model of the disease, could increase synapse number in these human cultures. Thus, the authors demonstrated the possibility of using an iPSCbased approach to gain an understanding of a complicated neural disorder and perhaps to screen for effective drugs.
Another good illustration of some of the points raised above is contained in a study on PD, a major neuro degenerative disorder affecting a subset of midbrain dopaminergic neurons [45]. Like ALS and AD, it is late onset and mostly sporadic, although a set of disease associated mutations has been identified, the most common of which is in the Leucine-rich repeat kinase-2 (LRKK2) gene. To model the disease, Nguyen and colleagues [45] derived iPSCs from patients with a LRKK2 mutation. They then followed standard protocols to produce neuronal cultures that were not pure, but did contain dopaminergic neurons that were physiologically active. By microarray analysis, the neurons were similar to those found in human fetal brain. Compared to neurons produced from control patient iPSCs, they had high levels of expression of oxidative stress genes. They also appeared to have a higher level of the protein α synuclein, which forms characteristic aggregates in PD. Further, they seemed to be more susceptible to various stressors, such as hydrogen peroxide and 6hydroxy dopamine. Thus, although relatively immature, these iPS derived neurons were capable of modeling some aspects of this late onset disease. Additional studies will be needed to see how completely they reproduce the disease phenotypes, how reproducible these changes are when larger numbers of iPSC lines are tested and how other types of PD patientderived iPSCs will behave.
Finally, a study illustrating many of the points raised in this review was published by Lee and colleagues [56]. These investigators were interested in familial dysauto nomia (FD), a genetic disorder associated with death of certain neural crestderived neurons in sensory and auto nomic ganglia. The disorder is associated with a mutation of the IκB kinase complex-associated protein (IKBKAP) gene, resulting in a splicing defect and reduced level of fulllength IKAP protein. Fibroblasts were obtained from one young girl with FD and used to prepare iPSCs that were then induced to differentiate into different types of cells. Neural crest precursors showed a particularly low level of intact IKAP protein and had clear defects in migra tion and neuronal differentiation. Lee and colleagues further showed that kinetin, a plant hormone known to be effective when tested on lymphoblastoid cells from an Rubin and Haston BMC Biology 2011, 9:42 http://www.biomedcentral.com/1741-7007/9/42 FD patient, had some corrective effects on the FD neural crest precursors. This sets the stage for a more comprehensive drug screen using iPSCderived cells.

Cardiovascular disease and drug toxicity testing
Several interesting studies relate to cardiac myocytes made from reprogrammed cells. Itzhaki et al. [57] pro duced cardiac myocytes from iPSCs isolated from patients that have a K + channel mutation found in congenital long QT syndrome (LQTS), a disorder associated with cardiac arrhythmias. The myocytes also had increased action potential duration, and the authors were able to screen different pharmacological agents to see which ones could correct the underlying electrophysiological defect. In another study, CarvajalVergara and colleagues [58] pursued a very interesting and presumably rare disorder known as LEOPARD syndrome. It is characterized most frequently by hypertrophic cardiomyopathy and is caused by mutations in the gene (PTPN11) that codes for the phosphatase SHP2. Interestingly, iPSCderived cardiac myocytes from patients were larger than those from controls, and the group has begun to dissect abnormal signaling within these cells that might be abrogated to ameliorate the disease phenotype. This is the kind of study, carried out in a rare disease background, that could contribute more generally to our understanding of other, more common types of cardiomyopathy.
The other widely discussed use for iPSC technology is in producing cardiac myocytes and hepatocytes to facilitate preclinical human testing of drug side effects. To date, much more progress has been made in produc ing cardiac cells. Braam and colleagues [59] carried out an early study using human ESCs as a source of human cardiac myocytes. They tested about 12 drugs that were already known either to affect or not to affect cardiac cells in patients. Similar activities were reproduced in the stem cell derived cultures. This is the beginning of toxicity studies that, in the future, should be done in the predictive sense: testing drugs on patientderived cardiac cells before it is known how they will affect the patients. It will be especially important to decide how many patients' cells need to be tested to establish a sufficiently Figure 3. A schematic diagram of a possible approach to using a stem cell-based system in a drug discovery campaign. The central idea is that the cells used for screening, efficacy and safety testing would be patient-derived. Also, lines of cells, potentially prepared from many patients, would be used for efficacy and safety testing in vitro prior to testing on those patients in vivo. It is hoped that this will increase the probability of clinical success.
Rubin and Haston BMC Biology 2011, 9:42 http://www.biomedcentral.com/1741-7007/9/42 accurate estimate of the likelihood that an individual drug will have cardiac toxicity and, even more importantly, to help to identify biomarkers for sensitive and insensitive patients.

Prospects for the future
There seems to be a widely held belief that the drug discovery system, as generally used in the pharmaceutical industry, needs to be improved, perhaps radically. In this article, we have suggested that a stem cellbased program might do just that by providing human diseaserelevant cells in numbers large enough to be used to: discover new pathologies, thereby establishing better drug targets; carry out more predictive primary and secondary screen ing assays; test drug safety; and identify subsets of patients most likely to respond to particular therapeutic classes. This is summarized in Figure 3.
Much work needs to be done before we can be certain that differentiated cells produced from patientderived iPSCs will offer any dramatic advantages. At present, we are uncertain whether the process of reprogramming, so essential to this method, is fundamentally flawed. Our view is that there will be many technical improvements over the next few years in the method of producing patientspecific differentiated cells (via reprogramming, transdifferentiation or partial reprogramming) to allow all of the relevant studies to be executed. A greater under standing will also be achieved with respect to the reproducibility of the process. At present, we do not know even how many iPSC clones per individual patient need to be produced to provide adequate consistency, nor do we know the true variability in response among cells derived from many patients with the same genetic disorder.
A final question that can be raised from the many types of in vitro studies described here relates to the seemingly ephemeral nature of the differentiated state. Why is it so relatively easy to change one type of cell into a radically different one? Does this suggest that cell identity could be changing, to at least some degree, much of the time? Does the existence of metaplastic cells also suggest the possibility that this phenomenon can occur without external manipulation? If so, does it further suggest the possibility that this process can be mobilized for thera peutic purposes? Will it be possible to interconvert cells using drugs -for instance, making muscle out of fat or connective tissue, or neurons out of glia -as an entirely different way of treating degenerative disorders or diseases of aging? If nothing else, new biological concepts derived from studying stem cell behavior may contribute to completely novel modes of treatment for serious diseases.