Introduction

New challenges for hazard and risk assessment in pharmaceutical and chemical industries and problematic animal testing require the development of novel in-vitro models for the molecular characterization of drug- or chemical-related effects. On the side of toxicity testing, in Europe REACH (Registration, Evaluation and Authorization of Chemicals) has passed the first hurdle to become legislation in November 2005 [1], and estimates of related costs and time lines for testing the approximately 30 000 eligible substances produced at quantities above 1 t in the European Union (EU), range from 2.4 to 8 billion Euros and 11 to 40 years, and are supposed to consume between 4 and 40 million test animals [2]. There are related projects in the US (voluntary EPA-High Production Volume Challenge program = HPV) [3] and Japan (TTREC = Toxicity Testing Reports for Environmental Chemicals) [4], and thus validated alternative test methods are urgently needed for this upcoming dramatic surge of safety tests. Whereas a number of animal tests for topical toxicity have been successfully replaced by alternative methods, systemic and in particular reproductive toxicity require essentially novel test strategies in order to achieve adequate safety levels [5].

On the other hand, next to toxicity, efficacy must be tested before compounds can be applied to patients in clinical phases. Related animal models for some of the most important areas of human disease, for example, cancer and neurodegeneration, require very laborious, sophisticated and often extremely disruptive, irksome and even cruel procedures. Complex functional, molecular and behavioral read-outs are often an absolute necessity for preclinical therapeutic trials. Some examples include xenograft models for various human cancers [6-8]; MCAO-models for stroke by induction of severe cerebral ischemia by permanent occlusion of the middle cerebral artery in a variety of species [9-11]; various transgenic rodent models for conditions such as Alzheimer's disease (AD) [12-15], or amyotrophic lateral sclerosis (ALS) [16], or extremely tough experimental models for autoimmune encephalomyelitis induced by vaccination oftest animals with the

Table 8.1 A compilation of current published protocols for differentiated endpoints from murine and human embryonic stem cells (ESC).a)

Germ layer Tissue

Cell type

Ref.

Murine embryonic stem cells

Ectoderm Skin

Melanocytes

[96]

Keratinocytes

[97]

Neural

Neurons (dopaminergic)

[98]

Neurons (dopaminergic)

[99]

Neurons (dopaminergic)

[100]

Neurons (dopaminergic)

[101]

Neurons

[102]

Neurons

[103]

Neurons/glial cells

[104]

Neurons/glial cells

[105]

Neurons/glial cells

[106]

Oligodendrocytes

[107]

Glial cells

[108]

Mesoderm Blood

Hematopoietic cells

[109]

Hematopoietic cells

[110]

Mast cells

[111]

Dendritic cells

[112]

Macrophages

[113]

Lymphoid precursors

[114]

Adipose tissue

Adipocytes

[115]

Endothelial

Vascular progenitors

[116]

Vascular progenitors

[117]

Cardiac muscle

Cardiomyocytes

[118]

Cardiomyocytes

[119]

Cardiomyocytes

[120]

Cardiomyocytes

[121]

Cardiomyocytes

[122]

Skeletal/smooth muscle

Muscle cells

[123]

Muscle cells

[124]

Smooth muscle cells

[125]

Cartilage

Chondrocytes

[126]

Bone

Osteoblasts

[127]

Endoderm Pancreas

Pancreatic cells

[128]

Pancreatic cells

[129]

Pancreatic cells

[130]

Liver

Hepatocytes

[131]

8.1 Introduction 207

Germ layer Tissue

Cell type

Ref.

Human embryonic stem cells

Ectoderm Skin

Keratinocytes

[132]

Melanocytes, keratinocytes

[133]

Neural

Motor neurons

[134]

Motor neurons

[135]

Neurons

[136]

Neurons

[137]

Neural precursors

[138]

Mesoderm Blood

Lymphohematopoietic cells

[139]

Hematopoietic cells

[140]

Hematopoietic cells

[141]

Hematopoietic cells

[136]

Cardiac muscle

Cardiomyocytes

[142]

Cardiomyocytes

[143]

Cardiomyocytes

[144]

Skeletal/smooth muscle

Muscle cells

[145]

Cartilage

Chondrocytes

[145]

Bone

Osteoblasts

[145]

Endothelium

Vascular progenitors

[146]

Endoderm Pancreas

Pancreatic cells

[147]

Pancreatic cells

[148]

Pancreatic cells

[149]

Pancreatic cells

[133]

Liver

Hepatocytes

[150]

Hepatocytes

[133]

a) The list may not be comprehensive, and it should be noted that, in most cases, the characterization of tissue-specific cell types mentioned has been performed on the level of markers. Only more recently have more studies additionally included functional read-outs. In this list, ESC derived from other organisms (e.g., Rhesus monkeys) are not included, essentially because rodents are the most widely used preclinical animals in various models, and human in-vitro systems would be much more desirable than any other in overcoming potential inter-species variations.

autoantigen myelin oligodendrocyte glycoprotein (MOG-EAE) [17-19] as preclinical models of multiple sclerosis (MS). However sophisticated these models are, there is a general sense of caution towards them, because often their read-outs are insufficient or misleading [20]. Taken together, in both areas of toxicity and efficacy testing, there is a need for the development of novel in-vitro methods, which can balance disadvantages in terms of organ-specific barriers and metabolism with advantages regarding the higher relevance of humanized systems, more precise functional and molecular read-outs, and the potential of higher throughput. Given emerging regulatory challenges, the purely diagnostic use of human embryonic stem cell (ESC) models appears to develop into a highly attractive alternative. ESC can be differentiated to organotypic cell cultures and serve as flexible substrate for a variety of functional molecular endpoints. Moreover, they are ideal for validation purposes using modern silencing technologies in a framework of genetically homogeneous differentiations to various "tissue"-like cell culture substrates such as neural cells, cardiomyocytes, various types of muscle cells, and adipocytes. An overview of currently available protocols for differentiations of ESC is provided in Table 8.1.

Indeed, in combination with cutting-edge analytical technologies, and in particular proteomics technologies, there is hope of obtaining surrogate biomarker information on the level of protein signatures, which by taking into account post-translational modifications, can serve as content for next generation, high-throughput screening (HTS) methods [21, 22]. These protein signatures also have the intrinsic potential of representing a comprehensive view of underlying mechanisms, including and relating key protein surrogates for mechanistic aspects (pathways and modes of action), toxicity-related events and/or others of purely descriptive diagnostic value [22, 23].

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