New Opportunities for Stem Cells in Drug Discovery and Development

Tue, 01/07/2014 - 12:53pm
Estelle Doudement and Hirdesh Uppal, Genentech Inc.

Compound screening in 96-well plate for differentiation of human embryonic stem cells into endoderm. The nuclei are stained blue, and SOX17 specifi c endoderm marker is stained green. (Source: Genentech)Stem cells carry promises for regenerative medicine and cell therapy, but are also changing the drug discovery and development process. Stem cell models offer new opportunities to improve the manner in which pharmaceutical companies identify lead candidates and bring new drugs to the market. In spite of promising applications, new competencies surrounding stem cell differentiation and proliferation, induction of pluripotent stem cells and creation of efficacy assays are needed to make successful use of stem cells in drug discovery. As part of Genentech’s mission on scientific innovation, our group is attempting novel and pioneering science with stem cells and cellular reprogramming. Our work focuses on using stem cells to establish human cellular models with better reproducibility and predictability, which, as a result, will improve detection of toxic compounds during drug development. From high-throughput screening to human disease models, to safety and toxicology testing, stem cells are becoming an integral part of the drug discovery toolbox. 

Beyond an improved in vitro model
Pharmaceutical and biotechnology companies have been looking for new tools to improve drug discovery. From clinical failures, which have cost the industry over $2 billion for liver toxicity alone in the last decade,1 to high drug attrition rates and adverse effects, the need to establish better predictive models and to improve efficiency of go/no-go decision making early on in drug development is critical. Animal models are currently the best relevant physiological systems to evaluate drug efficacy and toxicity, but cost, time and ethical issues associated with in vivo studies—as well as the uncertainty of translatability to humans—emphasize the advantages of relevant in vitro models. 

Reliance on cellular systems in drug development will only become standard if both the industry and the regulatory agencies are moving in the same direction. The U.S. Food and Drug Administration (FDA) is already placing added importance to in vitro testing, recommending the use of human cell lines to characterize drug metabolic pathways.1 Although immortalized cell lines have their advantages, they are not the best representation of normal tissue, so right now primary cells are gold standard for in vitro toxicity testing. Yet, human primary cells present significant challenges, including cost, limited or no accessibility for particular cell types—such as pneumocytes—short life span and loss of function in vitro, which prevents chronic toxicity studies.

In addition, limited availability of cells from one donor and donor-to-donor variability make high-throughput screening studies inconsistent. 

Emergence of stem cell technologies provides new opportunities to build innovative cellular models. Access to embryonic stem cells (ESCs) has improved over the last decade and is no longer a limiting factor. As of November 2013, more than 230 human embryonic stem cell lines were eligible for use in research funded by the National Institutes of Health. Capable of dividing indefinitely and differentiating into any cell type, ESCs can offer an unlimited and consistent source of cells for high-throughput screening at a reduced cost, be used for chronic studies and provide a wide range of cell types. Plus, efforts have been made to generate embryonic stem cells lines for multiple species including mouse,2 rat,3 cynomolgus monkey4 and dog,5 enabling cross-species comparisons and in vitro/in vivo correlations, which are highly valuable for drug development. 

Beyond improved models, pluripotent stem cells technologies are introducing applications that were previously not possible. Currently, human clinical populations are poorly represented in drug development with a lack of genetic heterogeneity in human cellular models and a limited number of human disease models. With the 2006 discovery of induced pluripotent stem cell (iPSC) technology,6 researchers can generate pluripotent stem cells from adult somatic cells, preserving the genetic information within those cells. As a result, new cellular models can be created from individuals with a diverse range of drug susceptibilities and resistances, offering the premise of a “clinical trial in a dish” in a field where a personalized medicine approach is becoming increasingly predominant. 

Overcoming the challenges
Hepatocytes derived from stem cells via the use of small molecules (Green: Calcium; Red: Mitochondria, Blue: Nuclei). (Source: Genentech)Although companies have put tremendous efforts into providing tools, such as ESC maintenance and differentiation media and other reagents like transfection kits or antibodies to help development of stem cells in drug discovery, many challenges—scientific and industrial—are yet to be overcome.

In theory, stem cells can generate any cell type, but the ability to differentiate ESCs into functional and differentiated cells has not been fully established. Overall, the field has focused on four main cell types: cardiac, hepatic, pancreatic and neuronal. For other cell types—such as lung and intestinal cells—differentiation technologies remain immature. Even for the predominantly differentiated cell types, differentiation strategies are not standardized and are often based on growth factors, making protocols expensive, poorly reproducible and limited in terms of scale-up. Moreover, they often require an extensive number of differentiation steps and still generate a heterogeneous population, preventing the use of these cellular models for specific organ or cell-type toxicity screening. So far, cardiac differentiation from stem cells seems to have made the most progress, while stem cell-derived hepatocytes still express immature markers, pancreatic cells continue to face low-yield differentiation, and neuronal differentiation still has difficulties in generating and distinguishing the numerous cell types.

Furthermore, since the core competencies are largely driven by academic research, pharmaceutical companies need to gain expertise in the technology. The pace of stem cell research—for example, a single differentiation experiment currently can take more than a month—is too slow to fit into timelines required by the industry. In addition, before pharmaceutical companies will invest in the development of such platforms, further demonstrations of success and potential applications are necessary. And last but not least, stem cell culture and differentiation need to be adapted to the high-throughput environment of drug discovery by developing standardized high-throughput and miniaturized assays for in vitro screening.

Nevertheless, some companies have already started to take on these challenges. At Genentech, our lab started by building a stem cell platform to gain expertise and explore differentiation applications. Efforts have been put to implement a standard, robust and easy-to-handle stem cell maintenance culture, a large banking of stem cells via cryopreservation, large-scale capabilities by adapting ESC culture in suspension, miniaturization of ESC culture for screening purposes and the development of innovative high-throughput assays for quality control.

Our novel approach to stem cell differentiation took advantage of Genentech’s expertise in developing specific inhibitors for biological targets. Using small molecules instead of traditional growth factors to differentiate stem cells makes protocols less expensive, more reproducible and scalable, which are amendable to industrial processes. Likewise, other research groups also have implemented small molecules strategies. For example, the Rho-associated protein kinase inhibitor is now commonly used to improve stem cell survival and to culture them as a single cell suspensions, simplifying their maintenance.7 Numerous small molecules have also been used to improve differentiation of stem cells into various cell types.8–10 Even a small molecule-enabled reprogramming technique to transform adult somatic cells into iPSCs has been discovered.11 At Genentech, by screening potent and specific inhibitors on stem cells, we discovered a small molecule that enables stem cell differentiation into a pure endoderm population that is suitable for drug discovery (manuscript in preparation). The optimized process has a limited number of growth factors, relies mainly on one small molecule that generates an almost pure endoderm population that can be maintained through numerous passages and can be further differentiated into cell types of interest in drug discovery, including hepatic, pancreatic and potentially lung and intestinal cells. Our protocol is streamlined, cost effective, reproducible, efficient and flexible enough for industrial applications, but, most importantly, it has resulted in the ability to produce a homogeneous, mature hepatic cell population that can be an alternative to primary hepatocytes for in vitro toxicity screening.

Improving differentiation strategies requires the development of high-throughput assays in parallel. At Genentech, we used our expertise in high-throughput screening systems to miniaturize the stem cell cultures and develop specific assays for quality control, such as expression of pluripotent markers, and for screening small molecules to replace growth factors, such as assays to detect specific lineage markers to characterize differentiated cells. We have developed these assays using various technology platforms—including immunoassays, high-content imaging and flow cytometry—to cross validate results. The high-throughput stem cell platform can now be used to

screen chemical compound libraries on stem cells—during the differentiation process and on differentiated cells—for chemical safety profile testing or for new target identification.

Stem cell research also brings new opportunities for novel technologies such as single-cell analysis, which can follow the differentiation of a single stem cell to a differentiated phenotype and study how a drug affects this process.12

Current and future applications
A toxicity assay shows the effects of amiodarone on stem cell-derived GE Healthcare Cytiva cardiomyocytes. (Source: Genentech)By gaining expertise in the field, pharmaceutical and biotech companies have found critical applications of stem cell technologies in their drug development processes. Since multiple cell types are now available and have been thoroughly characterized, stem cells and iPSC-derived cells are already being used for in vitro toxicity screening in many pharmaceutical companies.

Genentech is now routinely using ES cell and iPSC-derived cardiomyocytes from GE Healthcare and Cellular Dynamics International (CDI) as high-throughput models to assess cardiotoxicity for drugs in development as well as to investigate specific mechanistic cardiotoxicity findings during in vivo studies or in the clinic. Our efforts to innovate and implement new tools like microelectrode array (MEA) for electrophysiological mapping of cardiac cells are in line with the FDA’s 2004 Critical Path Initiative (CPI), which pointed out the need for better predictive models to study QT interval prolongation.

Although the discovery of iPSC technology is only a few years old, the development of disease models is already changing the paradigm of preclinical studies. iPSC-derived disease models have been generated mainly for monogenic diseases, including neurological disorders like Parkinson’s disease and Reet syndrome, blood diseases like Fanconi anaemia, cardiac syndromes like LEOPARD, pancreatic type 1 diabetes and hepatic disorders like alpha-1 antitrypsin deficiency.13 Not only is the adult somatic cell’s genetic information conserved in iPSCs, but it can also be maintained once the iPSCs is further differentiated into various cell types. As an example, Ebert et al. showed that iPSC-derived cells from spinal muscular atrophy (SMA) patients could be further differentiated into motor neurons, which displayed a decrease number of nuclear SMA structures called gems, a phenotype associated with the disease.14 Also, Byers et al. showed that Parkinson’s disease iPSCs differentiated into dopaminergic neurons exhibited early phenotypes linked to the disease.15 Finally, Itzhaki et al. used long QT syndrome patient iPSC-derived cardiomyocytes, which exhibited QT prolongation characteristic of the disease, as a model that successfully evaluated pharmaceutical drugs predicted to aggravate or ameliorate the disease.16 

Use of stem cells can also help to identify new targets. Very recently, two publications identified a new target for Parkinson’s disease for which no current drug exists. Chung et al. showed that the molecule originally discovered from a 200,000 compounds screening on yeast could reverse the damages in iPSC-derived neurons with alpha-synuclein mutations, associated with Parkinson’s disease.17,18  Companies like iPierian (South San Francisco, Calif.) aim to advance novel therapeutics into the clinic for neurodegenerative diseases by utilizing iPSC technology. 

The need to study the temporal progression of disease is gaining more attention. Primary neuronal cells from diseased individuals represent an endpoint of the disease and do not allow researchers to study the long-term degeneration process. Differentiation of iPSCs obtained from healthy or early stage neurodegenerative diseased patients into neuronal cells represents a possible tool to study the degeneration process. Stem cell differentiation can thus drive innovation in drug discovery via time-dependent stem cell differentiation studies. At Genentech, in order to support the development of a molecule, we used an innovative model of hematopoietic stem cell (HSCs) differentiation into megakaryocytes to investigate potential mechanisms for thrombocytopenia development with trastuzumab emtansine (T-DM1; Kadcyla), which was seen in a subset of patients during clinical trials.19 T-DM1 did not act directly on the production of platelets but acted by reducing the number and maturation of megakaryocytes formed from HSCs. Use of primary cells—either HSCs, megakaryocytes or platelets—separately would not have allowed us to uncover this mechanism, demonstrating how stem cell differentiation has opened up new opportunities in investigative toxicology.

In spite of various challenges, companies have already invested in stem cell research and have identified central applications, lifting the uncertainty of how successful stem cell technology could be in drug discovery. Moreover, new opportunities are emerging and future applications could quickly shift from early stage drug development to the clinical space where iPSC technology could be used on patients to study idiosyncratic drug effects and heterogeneity in drug responses. These advancements also provide the opportunity to test the response of clinical candidates on iPSC-differentiated cells from a large population of individuals, giving drug development companies the ability to perform in vitro clinical trials. 

Successful results of collaborations
At Genentech, researchers built a stem cell platform to gain expertise and explore differentiation applications. (Source: Genentech)The progression of utilization of stem cells in drug discovery will only be possible by collaborative research. Sharing expertise between academic research and industry, as well as investment from pharmaceutical companies in basic and translational stem cell research, are key to develop successful applications. Collaborations can also take place between pharmaceutical companies and research tool and technology companies to help develop and validate efficient tools and reagents for stem cells work. Research-based collaborations have also arisen between global healthcare companies and stem cell academic experts such as those between GE Healthcare and Geron,

GalxoSmithKline and The Harvard Stem Cell Institute or Roche and CDI. In addition, the nonprofit organization, the California Institute for Regenerative Medicine has helped advance the use of stem cell technologies in drug discovery and development by closely monitoring progress in stem cell research and by targeting funding in areas most likely to lead to new therapies.

Collectively, the great progress in the field of stem cell research in the industry, including our group’s work at Genentech in stem cell biology and toxicology, is expected to decrease the risk of late-stage failure of new chemical entities and large molecule drugs, reduce R&D costs and increase the likelihood and rate of bona fide drug discovery—all primary objectives in the pharmaceutical industry today. Importantly, the investment by the industry in stem cell biology and technologies is assisting to overcome the hurdles to full-scale industrial applications, to develop scientific leadership and innovation and to accelerate the drug development process.  


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15. Nguyen HN, Byers B, Cord B, et al. LRRK2 mutant iPSC-derived DA neurons demonstrate increased susceptibility to oxidative stress. Cell Stem Cell. 2011;8(3):267-80.

16. Itzhaki I, Maizels L, Huber I, et al. Modelling the long QT syndrome with induced pluripotent stem cells. Nature. 2011;471(7337):225-9.

17. Chung CY, Khurana V, Auluck PK, et al. Identification and rescue of alpha-synuclein toxicity in Parkinson patient-Derived Neurons. Science. 2013;342(6161):983-7.

18. Tardiff DF, Jui NT, Khurana V, et al. Yeast reveal a “Druggable” Rsp5/Nedd4 network that ameliorates alpha-synuclein toxicity in neurons. Science. 2013;342(6161):979-83.

19. Mahapatra K, Darbonne W, Bumbaca D, et al. T-DM1-induced thrombocytopenia results from impaired platelet production in a HER2-independent manner [abstract]. Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics, 2011 Nov. 12-16, San Francisco, Calif. Molecular Cancer Therapeutics. 2011;10(11 Suppl):A135.


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