Embryonic stem cells were first introduced into the scientific community in 1981 when they were isolated from mice, setting forth a cascade of paradigm shifting scientific events. More than a decade afterwards, in the late 1990s, human embryonic stem cells were isolated2 and for the first time, their potential seemed to be ‘limitless’. The ability of stem cells to become any cell type in the body offered great promise in regenerative medicine.

Particularly, in devastating diseases such as diabetes and neurological disorders, which are among and remain the hardest diseases to treat today. Stem cells also held the premise in drug discovery providing valuable information on toxicity and disease profiling. Still, after nearly two decades of experimentation, much remains unknown about stem cell fate and its future in personalised medicine.

During embryonic development, after the fertilisation process, meiosis occurs and results in a series of cleavage steps resulting in the morula. The morula is comprised of totipotent cells that give rise to the blastocyst. The inner cells of the blastocyst are pluripotent and give rise to stem cells that can differentiate into any cell type in the body, thus, their potential in a variety of diseases. The signalling mechanisms by which this occurs are very complex and demonstrate the enormity of interactions in stem cell pluripotency and differentiation. The potential of stem cells prompted an interest in understanding the differentiation and self-renewal properties and, as a result, many researchers turned to small molecules – for their pharmacological control and ease of use. The first small molecule found to play a role in differentiation of stem cells was retinoic acid3. In 1978 Retinoic acid sparked the interest and in 1998 the fire was ignited again when human embryonic stem cells were isolated4. The use of small molecules in determining stem cell fate became a fad demonstrated by the huge increase in publications since 1998 (Figure 1). Despite such an increase, there has yet to be a small molecule in development; and with the potential of entering the clinic to treat patients using stem cell therapy. As an example, retinoic acid, one of the oldest and most popular differentiating small molecules, has yet to demonstrate its value in the clinic. As the need to discover small molecule(s) that may have potential clinical use arose, many groups and companies turned their focus on large chemical library screening using innovative and complex high content assay approaches built on robust high-throughput screening (HTS) platforms5. Several high throughput screens have been conducted to date to identify small molecules that play a role in stem cell differentiation and self-renewal. Investigators were successful at finding only a handful of small molecules that played a role in differentiation. Many challenges were encountered by many and still surround the amenability and heterogeneity of stem cell assays in HTS. One of the biggest challenges is the disparity between embryonic stem cell models, ie mouse Embryonic Stem Cells (mESCs) versus human Embryonic Stem Cells (ESCs). Even with the latest techniques using nuclear transfer cloning and fusion with ES cells, the difficulties remain. Such difficulties, along with the lack of disease relevant models for many diseases, impelled the creation of induced pluripotent stem cells (iPSCs) with the ultimate goal to solve the discovered inherent problems with stem cells. The generation of iPSCs from mouse embryonic and adult fibroblasts was done by introducing four factors: Oct3/4, Sox2, Klf4, and c-Myc6. The introduction of iPSCs allowed many to work with disease relevant models and opened a whole new avenue in stem cell research. Not only do iPSCs provide a disease relevant model, iPSCs are readily amenable to HTS (Ramirez et al, unpublished observations). However, the introduction of the maintenance factors renders the cell models genomically unstable. Thus, one must question whether there is really a future for small molecules in personalised medicine based on either hESCs or iPSCs.

HTS of stem cells: an emerging catalogue of molecules
Although the concept of using hESCs in HTS is attractive to many, there are still many challenges that need to be overcome. These challenges include, but are not limited to: sufficient source material for the isolation, the use of a feeder layer for expansion, and spontaneous differentiation. As a result, many investigators have used mESCs as surrogates for hESCs.

Unlike the growth requirements and delicacy of hESCs, mESCs can be grown in the absence of feeder cells using leukaemia inhibitory factor (LIF)7; making them amenable to scale up for HTS. In addition to LIF, other growth factors, such as serum, are included in the media of the mESCs for long-term expansion. However, investigators have found that BMP4, which blocks differentiation, can be used to replace serum8. While the use of a human model is highly desirable by many investigators, the process of hESC isolation and expansion can be seen to many as being tedious and at times expensive. As a result, mESCs have been widely used in small molecule screening campaigns. In 2003, the first high-throughput screen run on mESCs led to the identification of TWS119, a small molecule found to induce neuronal differentiation in mESCs9. This screen set forth a series of cascading events resulting in a vast list of small molecules capable of differentiating neuronal cells, hematopoietic cells, cardiomyocytes and oesteocytes.

Since then, a number of small molecules have been shown to play a role in both hESC and mESC fates using HTS as well as other methods. These identified compounds are summarised in Table 1. Some of the small molecules shown to have an effect on the differentiation of stem cells also showed value in other areas. Such is the case for the anticancer drugs imatinib, bortezomib and geldanamycin. Imatinib, also known as Gleevec, is a well known BCR-ABL and PDGFR inhibitor and plays a role in cell viability, cell proliferation and apoptosis10. Similarly, bortezomib, a proteasome inhibitor, plays a role in apoptosis, cell viability, cell proliferation and cell adhesion11. With this in mind, one may come to the conclusion that the use of small molecules with known clinical use to treat cancer or other diseases may only cause further genomic instability; potentially leading to the development of additional unwanted cancers. While mESCs have proved useful in HTS looking for small molecules that exert an effect on stem cell fate, much has yet to be learned on the diversity of the different populations and the potential differences in the effects of the molecules by species.

Similarly, many investigators have purposely created genomic instability by introducing certain mutations in stem cell systems in order to model diseases. However, with the growing ethical dilemmas presented when using embryonic stem cells and the need to model diseases that do not have a particular phenotype associated with it, induced pluripotent stem cells (iPSCs) were developed and to date have been the most amenable stem cell type to HTS (Figure 2).

Induced pluripotent stem cells were introduced in 2007 when ‘stem cell-like’ human fibroblasts were derived using the factors Oct3/4, Sox2, Klf4, and c-Myc12. As a result, many used this to their advantage to model pathogenesis using viral vectors and plasmids to introduce such factors. In addition to fibroblasts, liver and epithelial cells have been reprogrammed into iPSCs13. Induced pluripotent stem cells have been generated as models for amyotrophic lateral sclerosis, Parkinson’s disease, spinal muscular atrophy and familial dysautonomia14-17. In addition to the modelling of diseases, iPSCs require less maintenance than embryonic stem cells. They lack the need of a feeder layer and can be expanded using laminin, fibronectin and polyornithine16. Furthermore, iPSCs can be used to model diseases in which there is no phenotypic change in the cells of the disease16. One of the major challenges of iPSCs is the introduction of such factors as c-myc that will ultimately cause genomic instability in the cells. With this in mind, one has to wonder whether iPSCs will ever be used for personalised methods. New methods using episomal vectors to reprogramme cells may shed some light in this avenue; however, much remains unknown about their use in reprogramming cells. As such, investigators are in search of small molecules that would reprogramme cells without the introduction of exogenous factors. As a result, further advancement in HTS of stem cells is needed.

Stem cell small molecule therapeutics
The therapeutics race to find a drug or a combination of drugs that further the potential of regenerative medicine continues. Small molecules can target signalling pathways, receptors, genes and mechanisms instrumental in the manipulation of stem cell fate. Although small molecules have been helpful in understanding the fundamental biology of stem cells, the utility in governing stem cell fate in vivo is far from being understood. There is a complexity of intrinsic and extrinsic factors at play that is only beginning to be teased out.

The development and discovery of small molecules have targeted three main biological properties of stem cells: their ability to differentiate, selfrenew and reprogramme. Self-renewal assays were first seen in mESCs while efforts were made to find small molecules that had an effect on the LIF or BMP signalling pathways. This led to the discovery of BIO and SB216763, found to inhibit GSK-318,19. Interestingly enough, while these small molecules communicated via similar signalling pathways, they not only exhibited selfrenewal properties but also demonstrated differentiation properties. This clearly demonstrates the ability of small molecules to affect stem cell fate not only across species but also within the cell itself.

With advances in high content screening, Oct4, a marker of pluripotency, has been pinned as a key player in the development of fluorescentbased assays to find small molecules promoting self-renewal. Using this method, SC1 was identified. Unexpectedly SC1, an inhibitor of both RasGAP and ERK1, lacked any association with the LIF or BMP signalling pathways while maintaining its ability to promote self renewal in embryonic stem cells20. In our lab using a similar method with Oct4, we were able to identify theanine, sinomenine, gatifloxacin and flurbiprofen as regulators of self-renewal in hESCs. Gatifloxacin, an antibiotic, was the only compound found to have an effect on mESCs among these compounds, demonstrating the vast differences between species21. Taken together, these small molecules have shown that several different signalling pathways appear to be involved in stem cell self-renewal. Thus, there is lack of selectivity within the compounds revealing the need to find signalling molecules that play a more centralised role in cell self-renewal.

Many are hopeful that the pluripotent stem cells will act as a resource for renewable cells and tissue with the potential to aid in treatment of diseases and injuries such as Parkinson’s disease, ALS, spinal cord injury and arthritis. A large number of small molecules have provided insight towards understanding the intricate system at play in the differentiation of embryonic stem cells; yet at present, it remains but a glimpse. They have targeted several pathways such as Wnt, Hedgehog and Notch, which are at the epicentre of developmental biology and therapeutic investigations. One of the key small molecules targeting the Wnt pathway is TWS119, the first small molecule identified via HTS. TWS119 is a GSK-3 inhibitor, which communicates via the Wnt signalling pathway and promotes the differentiation of embryonic cells to neuronal cells. Subsequently, countless discoveries of small molecules were made (Table 1). In addition to their role as anti-neoplastics, drugs such as imatinib and bortezomib have been shown to have differentiating potential. Bortezomib has been shown to induce osteoblast formation through activation of beta-catenin/TCF signalling. Imatinib, a signal transduction inhibitor, used to treat chronic myelogenous leukaemia (CML) has been shown to be useful in maintaining viability and differentiation potential in embryonic stem cells. In comparison to the other properties, small molecules affecting differentiation well outnumber those found to affect the self-renewal and reprogramming capabilities of embryonic stem cells. Thus small molecules are being praised for their differentiation abilities, yet their success in vivo has yet to be achieved.

Spontaneous differentiation of stem cells has been a great challenge to overcome. Most recently, Rho kinase inhibitors (ROCKi) have been found to preserve pluripotency in embryonic stem cells22 and have frequently been used to maintain selfrenewal in a variety of screens including genomewide RNAi screens23. However, the mechanisms by which ROCKi and other kinase inhibitor small molecules promote pluripotency are still poorly understood. The long-term effects of these kinase inhibitors remains a mystery and studies are needed to be certain genomic instability is not a consequence of using these small molecules.

We are far from the point of successful transfer of viable stem cells derived from small molecule altered embryonic stem cells. The challenges that lay ahead are largely encompassed by genomic instability and rejection. For this reason, the reprogramming capabilities of somatic cells have been exploited. The induction of pluripotent stem cells from adult fibroblast in 2006 has provided another potential platform for the creation of disease models and more personalised treatment. However, similar to the introduction of mutated genes in ES cells, the introductions of retroviral and lentiviral vectors cause genomic instability. Thus, investigators looked to HTS and complex cell-based assays to find small molecules that would reprogramme cells into iPS cells free of exogenous DNA. The small molecule reversine was found to be capable of such a feat9 and later confirmed24. More recent studies have shown that a combination of BIX-02194 and BayK8644 and kenpaullone can promote the expression of Klf4 and Sox2, respectively, factors necessary for reprogramming25,26. Still, further investigation is needed to determine the mechanism by which reprogramming occurs within the cell. At the present time, the transitional leap to the clinic appears in the very distant future; that is if we can overcome the cell-based screening hurdles and identify novel small molecules with desired pharmacological effects (Figure 3).

Conclusions
The processes of pluripotent cell maintenance and differentiation are complex and involve the expression of a series of signalling cascades at any given time. Thus, the notion that a small molecule could achieve what several signalling molecules have been shown to achieve is highly unlikely. To date, FDA-approved and marketed small molecule drugs target only 500 genes, the majority targeting enzymes and GPCRs27, whereas the genome constitutes approximately 20,000 genes. Therapeutic index is still a black box when it comes to small molecules. For this reason, 90% of drug candidates fail, mostly due to lack of efficacy. This begs the question whether the discovery of small molecules will ever have a significant impact on personalised medicine. The complexity of signalling mechanisms, the potential of causing genomic instability and the lack of genetic manipulation into viable cells has rendered the notion that small molecules will be able to be used in personalised medicine a mirage; a story that drummed much excitement at the beginning but its progression appears to be less and less exciting.


Dr Christina Ramirez is a former Assay Development Scientist at the HTS Core Facility who worked on assay implementation and screening of iPS cells. She is currently pursuing her PhD in cellular and molecular pharmacology at Rutgers University, USA. In 2008, she received her MS in biology from Seton Hall University, USA.

Dr Dana Duré is a former scientist at the HTS Core Facility. In 2012, she received her MD from SUNY Downstate Medical Center, USA. She is currently completing her residency training at SUNY with continued interest in cancer treatments at various stages of development and use.

Dr Hakim Djaballah, molecular pharmacologist and technologist, has been the Director of the HTS Core Facility at Memorial Sloan-Kettering Cancer Center since its establishment in 2003. In 1992, he received his PhD in biochemistry from the University of Leicester, England. He was the recipient of the 2007 Robots and Vision User Recognition Award.

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