WHITE PAPER

INTRODUCTION

Adult stem cells, embryonic stem cells, and induced pluripotent stem cells are cells that are capable of self-renewal and differentiation into different cell types.

These qualities make them potentially powerful tools for drug discovery, particularly in the fields of regenerative medicine, disease modelling, toxicity testing, gene targeting, and high throughput screening.

Progress in stem cell research has accelerated since the first successful stem cell therapy treatment in the 1950s using hematopoietic stem cells. However, the adoption of stem cells for drug discovery research by drug companies has been slow, mainly due to concerns with controlling stem cell differentiation, adequate supply of stem cells for high-throughput screening, and regulatory hurdles.

These issues are being addressed by research institutions, biotechnology companies, and drug companies. Thus, the future of stem cells in drug discovery is looking bright.

WHAT ARE STEM CELLS?

Stem cells are cells that serve as the foundation for all other types of cells in the human body. What sets stem cells apart from other cells are two important characteristics: they are capable of self-renewal through cell division, and they can differentiate into many different types of cells in vitro and in vivo.

In the human body, specialized cells such as red blood cells, heart cells, and brain cells all start out as stem cells. Stem cells can divide and multiply indefinitely either to remain as stem cells or differentiate into specialized cells. The ability to renew themselves perpetually have made stem cells an increasingly attractive area for research, particularly in the field of drug discovery.

TYPES OF STEM CELLS

There are three types of stem cells: embryonic stem cells (ESCs), adult or somatic stem cells, and induced pluripotent stem cells (iPSCs). Embryonic stem cells come from three to five-day old fertilized embryos. These embryos are products of in vitro fertilization that are given up for research with the consent of the donors who no longer need them. Embryonic stem cells are “pluripotent,” meaning they can divide indefinitely to produce more stem cells, or they can differentiate into any kind of specialized cells.

Adult stem cells, on the other hand, are found in limited numbers in many adult tissues such as the bone marrow and the lining of the stomach. Unlike embryonic stem cells, adult stem cells can only give rise to cell types specific to their tissue of origin.

For instance, hematopoietic stem cells, which are found in the bone marrow, can only develop into white blood cells, red blood cells, and platelets. Induced pluripotent stem cells are specialized adult cells that have been genetically reprogrammed to assume an embryonic stem cell-like state. These types of stem cells have the potential to reduce reliance on the controversial use of embryonic stem cells, and more importantly, could enable researchers to create tailor-made treatments for specific individuals by greatly reducing the chances of immune system rejection.

DISCOVERY/HISTORY OF STEM CELL USE

In the 1950s, scientists discovered that certain cells in the bone marrow could regenerate a leukemia patient’s proper blood functioning following chemotherapy and radiation. The reason for the transplanted bone marrow’s regenerative ability was uncovered in the 1960s, when scientists identified hematopoietic stem cells in the bone marrow that displayed two unique characteristics: the ability to self-renew and the ability to differentiate into all types of red and white blood cells. Bone marrow transplantation is the most successful form of stem cell therapy to date.

In 1981, the first ESCs were successfully derived from mouse blastocysts and in 1998 human ESCs were derived from fertilized embryos that had been donated for research. The controversy surrounding the use of human embryos to further stem cell research led scientists to find other ways to derive pluripotent stem cells. The big breakthrough came in 2006 when Japanese researchers reported the conversion of adult cells into an embryonic stem cell-like state, which they named induced pluripotent stem cells (iPSCs). These iPSCs were derived from mouse fibroblasts. By 2007, another breakthrough was made when adult human fibroblasts were successfully reprogrammed to iPSCs. iPSCs have since become the most promising of all stem cells in relation to drug discovery.

USES OF STEM CELLS IN DRUG DISCOVERY

Currently, the most advanced application for stem cells is in the study of cardiovascular and neurodegenerative diseases. Stem cells that have been programmed into either cardiac or neural cell types are being widely used in research on drug discovery and toxicity testing. Stem cells have become integral to the study of the molecular pathways that cause certain diseases as they progress. Furthermore, ESCs and iPSCs can be engineered to carry mutations to elucidate disease development. Analytical Biological Services Inc. White Paper

REGENERATIVE MEDICINE

Certain cardiovascular, musculoskeletal, and neurological diseases are not easily reparable. Stem cells could hold the key in the repair or regeneration of tissues and organs damaged by disease or injury. But many clinical challenges need to be overcome before stem cells become an acceptable treatment option. There are four major points to consider in using stem cells in regenerative medicine:

1. which stem cell can be induced to develop into a functional tissue;
2. designing appropriate scaffolds to grow the tissue on;
3. what role bioreactive molecules play in the formation of the target tissue;
4. studies on safety and grafting procedures. A more serious concern is the tumorigenic potential of stem cells caused by uncontrolled differentiation

Bone marrow transplantation is the earliest application of stem cells in regenerative medicine. It is also the only established treatment based on stem cells so far. Most of the potential applications for stem cells are still at various stages of experimentation. However, interest in the utilization of stem cells have made them a very attractive area for regenerative research and development. There are now more than 3,000 clinical trials of stem cells in progress (https://clinicaltrials.gov/).

DISEASE MODELLING

Understanding how human disorders develop is essential in formulating treatment methods. The use of animal models, tumor cells, and immortalized cells from tissue biopsies have presented

limitations on drug discovery for many diseases. For instance, the use of mice to model human disease progression can lead to incompatible results during clinical trials owing to the many genetic, developmental, and physiological differences between the two species.

Tumor cells and immortalized cells are too specific to their cell types and are difficult to sustain or manipulate during culture. Pluripotent stem cells (both ESCs and iPSCs) can overcome these limitations. The cells can be engineered to carry genetic defect(s) of interest and used to identify potential phenotypes at either the undifferentiated or differentiated states or both.

Different types of methodologies can be employed to elucidate the causes of specific diseases, leading to the design of unique therapies through high-throughput drug screening. Furthermore, patient-specific iPSCs could improve the identification of drugs that are effective for a specific population of patients.

TOXICOLOGY

Increasing exposure to chemical hazards in the environment has made toxicology an important study for reproductive and developmental biology. Indeed, many environmental factors that cause birth defects can be attributed to chemical exposure. The list of potentially hazardous chemicals being produced worldwide is expanding. In the United States alone, there are well over 80,000 registered chemicals for various commercial applications. However, data on toxicity and exposure to these chemicals are very limited.

Currently, pregnant mice, rats, or rabbits are employed to determine the teratogenicity of various chemicals, the results of which are used as guidelines for human exposure. However, use of these animal models is both expensive and time-consuming. It is also impossible to conduct high-throughput screening to study hundreds of potential toxicants at a time.

Pluripotent stem cells (ESCs and iPSCs) are alternative in vitro toxicology models that have tremendous potential to overcome many of the limitations posed by animal testing. Because these cells can differentiate into any cell type, they can be used to study the effects of suspected developmental toxicants.

Methodological advances to induce spontaneous and directed differentiation of PSCs together with advanced techniques in metabolomics, transcriptomics, proteomics, epigenetics, and high-throughput assay methods have resulted in an explosion of research in the field of stem cell toxicology. Mouse ESCs have been in use since the early 2000s for toxicity testing and various assays are now available to study toxicants.

Progress in the utilization of human ESCs has been slow due to legal and ethical concerns. But as with disease modelling, toxicity studies on human ESCs are more advantageous than mouse ESCs to eliminate species bias. The use of iPSCs for toxicity testing has so far been limited because of the documented lineage bias towards their origin, meaning they are not completely pluripotent. However, this could be the major advantage of iPSCs. Developing personalized toxicological assays can result in greater information on genetic diversity.

GENE TARGETING

Gene targeting is a technique wherein an endogenous allele of a target gene is exchanged for a mutated copy via homologous recombination. It was first successfully done in mouse embryonic stem cells in the late 1990s. Since then, this technology has allowed unmatched precision not only for how genes are manipulated but also to study the effect of such manipulations on the developing animal. Mouse ESCs are now used extensively in gene targeting studies as these cells have made it possible to alter the germ-line of mice in a predictable manner. Thus, gene targeting has enabled researchers to generate strains of mice with specific mutations in their genome

allowing the analysis of gene function in vivo. These “knockout” mice continue to provide enormous insight into mammalian development genetics. ESCs are essential for the success of gene targeting because they can be grown in large numbers. This makes them useful for introducing gene alterations, screening very rare homologous recombination events, and producing various types of animal models with designed mutations. In the future, gene targeting in ESCs is expected to throw light on complex pathways that are regulatory, metabolic, and physiological in nature.

HIGH-THROUGHPUT DRUG SCREENING

Underlying the success of stem cells in drug discovery and toxicity screening is their employability for large-scale culture conditions and screening formats. High-throughput screening (HTS) using small molecule drugs in human iPSCs for the purpose of identifying novel therapeutic compounds is only just beginning to influence the drug discovery process. By using stem cells, hundreds to thousands of small-molecule drug compounds can be screened for their efficacy or toxicity. High- throughput screening allows for the investigation of drug response at the genotype level in a more systematic and controlled manner. It can also be used to study methodologies for controlling the fate of stem cells using cost-effective small molecules. The challenge for HTS is how to obtain large numbers of stems cells consistently so that drug screening can proceed unhampered.

High-throughput screening is also increasingly being used in conjunction with high-content screening (HCS), which uses image analysis to quantify changes in cellular phenotype. The use of both methods makes it possible to investigate the genotype-phenotype-drug response relationship. In the past decade, HCS has generated large drug libraries using a combination of powerful microscopic, robotic, and computerized screening systems.

 

Current Challenges

STEM CELL DIFFERENTIATION

A major challenge for employing stem cells in many areas of research is the difficulty in controlling their differentiation. Stem cells can either spontaneously differentiate or be directed to differentiate into specific types of cells. When grown without differentiation inhibitors, ESCs and iPSCs will spontaneously differentiate in culture into identifiable embryonic structures. For instance, ESCs have been found to differentiate into embryonic endoderm and ectoderm, yolk sac, nerve cells, epithelial cells, tubular structures, cartilage, heart muscle, and blood islets.

Controlled differentiation is more technically challenging especially when large numbers of cells are required for HTS. Differentiation of stem cells to a specific type of mature cells normally requires serial cell culture steps with sequential addition of growth and patterning factors that essentially mimic in vivo processes during their development. In addition, the microenvironment in which stem cells are cultured also needs to be manipulated. Finally, all these conditions need to be reproducible and standardized for large-scale applications, especially in drug discovery. Thus, innovative techniques need to be developed for stem cells to have wider applicability in drug discovery.

SUPPLY

As noted earlier, the challenges currently being faced in producing large numbers of stem cells for research in general, and drug discovery, have resulted in a limited adoption of stem cells by pharmaceutical companies. Until recently, research institutions (both public and private) were the main sources of stem cells. ABS now provides a variety of virus-free and oncogene- free iPS cells. These include human adipose mesenchymal stem cells, human multipotent stem cells, CD34+ cord blood stem cells, and others. Please inquire for more information.

REGULATION

The International Society for Stem Cell Research released its latest version of the Guidelines for Stem Cell Research and Clinical Translation in 2016. The guidelines were created to set high standards for the review and conduct of research and clinical development in stem cell science worldwide. Furthermore, regulatory agencies exist in the European Union (European Medicines Agency), the United States (FDA), the United Kingdom (HFEA and HTA), and Japan (PMDA) that closely monitor all research involving stem cells, particularly those geared towards the development of therapies.

Many pharmaceutical companies remain wary of using stem cells for drug discovery. In the US, the use of such a novel technology as a substitute for well-established and FDA approved testing methods is not an easy change to make. Pharmaceutical companies are also uncertain of whether regulatory agencies will accept data derived from stem cell screenings. But the drug industry is slowly making the change. Some of the large pharmaceutical companies, such as Roche, Pfizer, and AstraZeneca have already formed partnerships with academic institutions and hospitals to use stem cells to screen drugs against certain diseases and conduct clinical trials.

CONCLUSION

Stem cells have unique characteristics that make them potentially significant tools for drug discovery. They have been employed in toxicity testing and gene targeting for over a decade, whereas their application in disease modelling, regenerative medicine, and high-throughput sequencing are still in the experimental or clinical trial phase. Progress in methodologies as well as in regulations continue to foster the use stem cells in drug discovery and as a replacement for many animal models.

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