Tumorigenicity assessment of cell therapy products: The need for global consensus and points to consider.

Pluripotent stem cells offer the potential for an unlimited source for cell therapy products. However, there is concern regarding the tumorigenicity of these products in humans, mainly due to the possible unintended contamination of undifferentiated cells or transformed cells. Because of the complex nature of these new therapies and the lack of a globally accepted consensus on the strategy for tumorigenicity evaluation, a case-by-case approach is recommended for the risk assessment of each cell therapy product. In general, therapeutic products need to be qualified using available technologies, which ideally should be fully validated. In such circumstances, the developers of cell therapy products may have conducted various tumorigenicity tests and consulted with regulators in respective countries. Here, we critically review currently available in vivo and in vitro testing methods for tumorigenicity evaluation against expectations in international regulatory guidelines. We discuss the value of those approaches, in particular the limitations of in vivo methods, and comment on challenges and future directions. In addition, we note the need for an internationally harmonized procedure for tumorigenicity assessment of cell therapy products from both regulatory and technological perspectives.

5-HT systems and anxiety: multiple mechanisms in the elevated X-maze.

Although much evidence supports the proposal that 5-HT neurones promote anxiety, there is also substantial evidence that the actual situation is more complex. The recent idea that 5-HT neurones, alerted by the forebrain, also function to restrain more primitive brainstem mechanisms has additional explanatory power but neither proposal accounts for the lack of responsiveness of 5-HT neurones to aversive stimulation from the environment. Evidence from the elevated X-maze anxiety model indicates that it is sensitive to multiple anxiety mechanisms which are not fully accounted for by either hypothesis. In addition, findings presented here indicate that open arm preference, risk assessment and overall motility load on separate factors in a factor analysis and that the anxiogenic action of 8-OH-DPAT under control conditions is a selective effect on open arm preference. This anxiogenic effect can be switched to anxiolytic at will by simple changes in experimental conditions. The outcome of 5-HT system manipulations on experimental anxiety may be the result of unstable balance between effects on different brain areas. It is suggested that 5-HT neurones do not carry information about threats in the environment, instead, this information is input locally where it modulates 5-HT release; this released 5-HT can have different consequences in different brain areas.

Effects of two stressors on behaviour in the elevated X-maze: preliminary investigation of their interaction with 8-OH-DPAT.

Effects of water deprivation and restraint were compared in the rat elevated X-maze. Water deprivation for 12-48 h increased corticosterone and had a duration-dependent "anxiolytic" effect in the elevated X-maze, increasing the ratio of open/total arm entries (OTR) and the proportion of time spent on the open arms (% time) without affecting total entries. Brain 5HIAA/5HT was increased only after 24 or 48 h deprivation. Restraint for 15 min also increased plasma corticosterone and brain 5HIAA/5HT but had no effect on behaviour in the elevated X-maze when rats were tested immediately afterwards. However, 1 h restraint was "anxiogenic" in the elevated X-maze immediately after release, reducing OTR and % time, but with a less consistent reduction in total entries; reductions in OTR and % time were still present 24 h later. The 5HT1A agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT) (0.1-0.2 mg/kg), administered 10 min before testing in the elevated X-maze, had "anxiogenic" actions in non-stressed rats. The effect of 0.1 mg/kg 8-OH-DPAT was not significantly altered by 24 or 48 h water deprivation but was abolished by restraint for 1 h immediately beforehand, despite the "anxiogenic" effect of restraint alone. Similar mutual antagonism of 8-OH-DPAT and restraint occurred when the dose of 8-OH-DPAT was increased to 0.2 mg/kg. Twenty-four hours after restraint, restrained rats which had received 8-OH-DPAT (0.1-0.2 mg/kg) still did not show any significant "anxiogenic effect" compared with non-restrained vehicle treated controls. Restraint-induced deficits in elevated X-maze exploration may prove a useful model with which to study the pharmacology of depression-related anxiety. However, the effects of the stressors examined, and their interaction with 8-OH-DPAT in the elevated X-maze, appear to depend on the nature of the stressor.

Multiple serotonin mechanisms in animal models of anxiety: environmental, emotional and cognitive factors.

Responses to serotonergic drugs in animal models of 'anxiety' are reviewed with emphasis on the elevated X-maze. Evidence for the 'classic' hypothesis, that decreasing serotonergic function is anxiolytic and increasing it anxiogenic, is most consistent in models of behavioural inhibition where the stimulus inhibits an approach response (conflict models). However, paradoxical drug effects are also frequent, especially where the aversive stimulus evokes an active response. Both types of drug effect are equally frequent in the elevated X-maze. 'Anxiety' models may detect multiple sites and mechanisms of action of the same drug; this may indicate multiple anxiety-related neurological mechanisms in the brain. However, not all drug effects in 'anxiety' models are necessarily related to anxiety itself. It is possible that cognitive factors may affect stimulus evaluation, and response inhibition by an aversive stimulus may be a special case of a wider role for serotonin in behavioural control. Clinical implications of these observations are considered.

An assessment of the elevated X-maze for studying anxiety and anxiety-modulating drugs.

The elevated X-maze has strong claims to validity as an animal model of anxiety, both in theoretical basis and drug responses. The model is sensitive to actual and putative anxiolytics, but because insufficient time has elapsed since its discovery, no agent first predicted to be anxiolytic in the elevated X-maze has been brought into general use yet. It has an advantage in detecting anxiolytic and anxiogenic agents under the same operating conditions. The design and execution of experiments with the model is discussed and it is shown that baseline arm preference and the size or direction of drug effects differ in the procedural factors affecting them. Because it presents features of both passive and active avoidance and approach/avoidance conflict, it may prove able to detect drug effects in different forms of human anxiety and aid in understanding their neurobiology.

Serotonin mechanisms in animal models of anxiety.

1. Responses to serotonergic drugs in animal models of anxiety are reviewed. Pre- and postsynaptic mechanisms and multiple sites of postsynaptic action contribute to conflicting findings. 2. Paradoxical responses to both serotonergic and non-serotonergic agents support the concept of multiple anxiety mechanisms. Non-anxiety factors, such as effects on cognition and behavioral inhibition, must also be taken into account. 3. Immediate 'anxiogenic' and delayed 'anxiolytic' effects most closely mimic the clinical effects of recently introduced anxiolytic drugs such as the selective serotonin reuptake inhibitors and buspirone. Thus the relevance to anxiety of immediate 'anxiolytic' effects of such agents in animal models is in question.

5HT drugs in animal models of anxiety.

It has been widely accepted that 5HT neurones promote anxiety, in humans as well as in animal models. This could be termed the "classic" hypothesis and it has led to a determined search for drugs which reduce 5HT function, especially agents which have selective actions at 5HT receptor subtypes. However, these novel agents tend to have weak and/or variable effects in animal models and more detailed examination of their actions suggests that not all findings are accounted for by the classic hypothesis. There appear to be circumstances in which increased 5HT activity can reduce anxious behaviour. There is increasing evidence for multiple anxiety mechanisms, which may be able to explain differential patterns of drug effects within and between models. Animal models of anxiety may also detect non-anxiety factors: effects on cognition or on impulsivity could be reflected in some models. This could be important in the light of recent evidence that 5HT-selective reuptake inhibitors are effective in impulsivity disorders. The classic hypothesis of 5HT function in anxiety may be only one part of an increasingly complex story. Unravelling the rest of this story is likely to lead to new insights in our understanding of anxiety and related disorders.

Serotonin mechanisms in animal models of anxiety

1. Responses to serotonergic drugs in animal models of anxiety are reviewed. Pre- and postsynaptic mechanisms and multiple sites of postsynaptic action contribute to conflicting findings. 2. Paradoxical responses to both serotonergic and non-serotonergic agents support the concept of multiple anxiety mechanisms. Non-anxiety factors, such as effects on cognition and behavioral inhibition, must also be taken into account. 3. Immediate 'anxiogenic' and delayed 'anxiolytic' effects most closely mimic the clinical effects of recently introduced anxiolytic drugs such as the selective serotonin reuptake inhibitors and buspirone. Thus the relevance to anxiety of immediate 'anxiolytic' effects of such agents in animal models is in question