Colture neuronali da cellule staminali in ambito farmacologico e tossicologico

[Smith I, Silveirinha V, Stein JL, de la Torre-Ubieta L, Farrimond JA, Williamson EM, Whalley BJ. Human neural stem cell-derived cultures in three-dimensional substrates form spontaneously functional neuronal networks. J Tissue Eng Regen Med. 2015 Feb 25.]

Abstract:

Differentiated human neural stem cells were cultured in an inert three-dimensional (3D) scaffold and, unlike two-dimensional (2D) but otherwise comparable monolayer cultures, formed spontaneously active, functional neuronal networks that responded reproducibly and predictably to conventional pharmacological treatments to reveal functional, glutamatergic synapses. Immunocytochemical and electron microscopy analysis revealed a neuronal and glial population, where markers of neuronal maturity were observed in the former. Oligonucleotide microarray analysis revealed substantial differences in gene expression conferred by culturing in a 3D vs a 2D environment. Notable and numerous differences were seen in genes coding for neuronal function, the extracellular matrix and cytoskeleton. In addition to producing functional networks, differentiated human neural stem cells grown in inert scaffolds offer several significant advantages over conventional 2D monolayers. These advantages include cost savings and improved physiological relevance, which make them better suited for use in the pharmacological and toxicological assays required for development of stem cell-based treatments and the reduction of animal use in medical research.

 

Annunci

I difetti e i danni all’Uomo della sperimentazione animale

[Akhtar A. The flaws and human harms of animal experimentation. Camb Q Healthc Ethics. 2015 Oct;24(4):407-19.]

Full Text: http://journals.cambridge.org/action/displayFulltext?type=6&fid=9949938&jid=CQH&volumeId=24&issueId=04&aid=9949937&bodyId=&membershipNumber=&societyETOCSession=&fulltextType=RA&fileId=S0963180115000079

Abstract:

Nonhuman animal (“animal”) experimentation is typically defended by arguments that it is reliable, that animals provide sufficiently good models of human biology and diseases to yield relevant information, and that, consequently, its use provides major human health benefits. I demonstrate that a growing body of scientific literature critically assessing the validity of animal experimentation generally (and animal modeling specifically) raises important concerns about its reliability and predictive value for human outcomes and for understanding human physiology. The unreliability of animal experimentation across a wide range of areas undermines scientific arguments in favor of the practice. Additionally, I show how animal experimentation often significantly harms humans through misleading safety studies, potential abandonment of effective therapeutics, and direction of resources away from more effective testing methods. The resulting evidence suggests that the collective harms and costs to humans from animal experimentation outweigh potential benefits and that resources would be better invested in developing human-based testing methods.

Nel testo:

Wide differences have also become apparent in the regulation of the same genes, a point that is readily seen when observing differences between human and mouse livers. 48 Consistent phenotypes (observable physical or biochemical characteristics) are rarely obtained by modification of the same gene, even among different strains of mice. 49 Gene regulation can substantially differ among species and may be as important as the presence or absence of a specific gene. Despite the high degree of genome conservation, there are critical differences in the order and function of genes among species. To use an analogy: as pianos have the same keys, humans and other animals share (largely) the same genes. Where we mostly differ is in the way the genes or keys are expressed. For example, if we play the keys in a certain order, we hear Chopin; in a different order, we hear Ray Charles; and in yet a different order, it’s Jerry Lee Lewis. In other words, the same keys or genes are expressed, but their different orders result in markedly different outcomes.

Recognizing the inherent genetic differences among species as a barrier to translation, researches have expressed considerable enthusiasm for genetically modified (GM) animals, including transgenic mice models, wherein human genes are inserted into the mouse genome. However, if a human gene is expressed in mice, it will likely function differently from the way it functions in humans, being affected by physiological mechanisms that are unique in mice. For example, a crucial protein that controls blood sugar in humans is missing in mice. 50 When the human gene that makes this protein was expressed in genetically altered mice, it had the opposite effect from that in humans: it caused loss of blood sugar control in mice. Use of GM mice has failed to successfully model human diseases and to translate into clinical benefit across many disease categories. 51 Perhaps the primary reason why GM animals are unlikely to be much more successful than other animal models in translational medicine is the fact that the “humanized” or altered genes are still in nonhuman animals.

In many instances, nonhuman primates (NHPs) are used instead of mice or other animals, with the expectation that NHPs will better mimic human results. However, there have been sufficient failures in translation to undermine this optimism. For example, NHP models have failed to reproduce key features of Parkinson’s disease, both in function and in pathology. 52 Several therapies that appeared promising in both NHPs and rat models of Parkinson’s disease showed disappointing results in humans.53 The campaign to prescribe hormone replacement therapy (HRT) in millions of women to prevent cardiovascular disease was based in large part on experiments on NHPs. HRT is now known to increase the risk of these diseases in women. 54

e:

“Appreciation of differences” and “caution” about extrapolating results from animals to humans are now almost universally recommended. But, in practice, how does one take into account differences in drug metabolism, genetics, expression of diseases, anatomy, influences of laboratory environments, and species- and strain-specific physiologic mechanisms—and, in view of these differences, discern what is applicable to humans and what is not? If we cannot determine which physiological mechanisms in which species and strains of species are applicable to humans (even setting aside the complicating factors of different caging systems and types of flooring), the usefulness of the experiments must be questioned.

It has been argued that some information obtained from animal experiments is better than no information. 64 This thesis neglects how misleading information can be worse than no information from animal tests. The use of nonpredictive animal experiments can cause human suffering in at least two ways: (1) by producing misleading safety and efficacy data and (2) by causing potential abandonment of useful medical treatments and misdirecting resources away from more effective testing methods.

Humans are harmed because of misleading animal testing results. Imprecise results from animal experiments may result in clinical trials of biologically faulty or even harmful substances, thereby exposing patients to unnecessary risk and wasting scarce research resources. 65 Animal toxicity studies are poor predictors of toxic effects of drugs in humans. 66 As seen in some of the preceding examples (in particular, stroke, HRT, and TGN1412), humans have been significantly harmed because investigators were misled by the safety and efficacy profile of a new drug based on animal experiments. 67 Clinical trial volunteers are thus provided with raised hopes and a false sense of security because of a misguided confidence in efficacy and safety testing using animals.

An equal if indirect source of human suffering is the opportunity cost of abandoning promising drugs because of misleading animal tests. 68 As candidate drugs generally proceed down the development pipeline and to human testing based largely on successful results in animals 69 (i.e., positive efficacy and negative adverse effects), drugs are sometimes not further developed due to unsuccessful results in animals (i.e., negative efficacy and/or positive adverse effects). Because much pharmaceutical company preclinical data are proprietary and thus publicly unavailable, it is difficult to know the number of missed opportunities due to misleading animal experiments. However, of every 5,000–10,000 potential drugs investigated, only about 5 proceed to Phase 1 clinical trials. 70 Potential therapeutics may be abandoned because of results in animal tests that do not apply to humans. 71 Treatments that fail to work or show some adverse effect in animals because of species-specific influences may be abandoned in preclinical testing even if they may have proved effective and safe in humans if allowed to continue through the drug development pipeline.

Modello “on a chip” di barriera placentare

[Lee JS, Romero R, Han YM, Kim HC, Kim CJ, Hong JS, Huh D. Placenta-on-a-chip: a novel platform to study the biology of the human placenta. J Matern Fetal Neonatal Med. 2015 Jun 15:1-9.]

Full Text: http://informahealthcare.com/doi/full/10.3109/14767058.2015.1038518

Abstract:

“Objective: Studying the biology of the human placenta represents a major experimental challenge. Although conventional cell culture techniques have been used to study different types of placenta-derived cells, current in vitro models have limitations in recapitulating organ-specific structure and key physiological functions of the placenta. Here we demonstrate that it is possible to leverage microfluidic and microfabrication technologies to develop a microengineered biomimetic model that replicates the architecture and function of the placenta.

Materials and methods: A “Placenta-on-a-Chip” microdevice was created by using a set of soft elastomer-based microfabrication techniques known as soft lithography. This microsystem consisted of two polydimethylsiloxane (PDMS) microfluidic channels separated by a thin extracellular matrix (ECM) membrane. To reproduce the placental barrier in this model, human trophoblasts (JEG-3) and human umbilical vein endothelial cells (HUVECs) were seeded onto the opposite sides of the ECM membrane and cultured under dynamic flow conditions to form confluent epithelial and endothelial layers in close apposition. We tested the physiological function of the microengineered placental barrier by measuring glucose transport across the trophoblast-endothelial interface over time. The permeability of the barrier study was analyzed and compared to that obtained from acellular devices and additional control groups that contained epithelial or endothelial layers alone.

Results: Our microfluidic cell culture system provided a tightly controlled fluidic environment conducive to the proliferation and maintenance of JEG-3 trophoblasts and HUVECs on the ECM scaffold. Prolonged culture in this model produced confluent cellular monolayers on the intervening membrane that together formed the placental barrier. This in vivo-like microarchitecture was also critical for creating a physiologically relevant effective barrier to glucose transport. Quantitative investigation of barrier function was conducted by calculating permeability coefficients and metabolic rates in varying conditions of barrier structure. The rates of glucose transport and metabolism were consistent with previously reported in vivo observations.

Conclusion: The “Placenta-on-a-Chip” microdevice described herein provides new opportunities to simulate and analyze critical physiological responses of the placental barrier. This system may be used to address the major limitations of existing placenta model systems and serve to enable research platforms for reproductive biology and medicine.”

Modelli di disturbi del neurosviluppo (come i disturbi dello spettro autistico) usando cellule staminali

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[Chailangkarn T, Acab A, Muotri AR. Modeling neurodevelopmental disorders using human neurons. Curr Opin Neurobiol. 2012 Oct;22(5):785-90.]

Full Text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3587787/

Abstract:

“The cellular and molecular mechanisms of neurodevelopmental conditions such as autism spectrum disorders have been studied intensively for decades. The unavailability of live patient neurons for research, however, has represented a major obstacle in the elucidation of the disease etiologies. Recently, the development of induced pluripotent stem cell (iPSC) technology allows for the generation of human neurons from somatic cells of patients. We review ongoing studies using iPSCs as an approach to model neurodevelopmental disorders, the promise and caveats of this technique and its potential for drug screening. The reproducible findings of relevant phenotypes in Rett syndrome iPSC-derived neurons suggest that iPSC technology offers a novel and unique opportunity for the understanding of and the development of therapeutics for other autism spectrum disorders.

Nel testo:

“Finally, animal models often do not recapitulate complex human diseases, and have been particularly problematic in the case of human neurodevelopmental disease such as autism.

Modello di sviluppo neurale e trattamento della sindrome di Rett usando cellule staminali

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[Marchetto MC, Carromeu C, Acab A, Yu D, Yeo GW, Mu Y, Chen G, Gage FH, Muotri AR. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell. 2010 Nov 12;143(4):527-39.]

Full Text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3003590/

Abstract:

“Autism spectrum disorders (ASD) are complex neurodevelopmental diseases in which different combinations of genetic mutations may contribute to the phenotype. Using Rett syndrome (RTT) as an ASD genetic model, we developed a culture system using induced pluripotent stem cells (iPSCs) from RTT patients’ fibroblasts. RTT patients’ iPSCs are able to undergo X-inactivation and generate functional neurons. Neurons derived from RTT-iPSCs had fewer synapses, reduced spine density, smaller soma size, altered calcium signaling and electrophysiological defects when compared to controls. Our data uncovered early alterations in developing human RTT neurons. Finally, we used RTT neurons to test the effects of drugs in rescuing synaptic defects. Our data provide evidence of an unexplored developmental window, before disease onset, in RTT syndrome where potential therapies could be successfully employed. Our model recapitulates early stages of a human neurodevelopmental disease and represents a promising cellular tool for drug screening, diagnosis and personalized treatment.”

Modello di muscolo umano per valutare la reazione clinica a farmaci e simulare malattie muscolari

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[Madden L, Juhas M, Kraus WE, Truskey GA, Bursac N. Bioengineered human myobundles mimic clinical responses of skeletal muscle to drugs. Elife. 2015 Jan 9;4:e04885.]

Full Text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4337710/

Abstract:

Existing in vitro models of human skeletal muscle cannot recapitulate the organization and function of native muscle, limiting their use in physiological and pharmacological studies. Here, we demonstrate engineering of electrically and chemically responsive, contractile human muscle tissues (‘myobundles’) using primary myogenic cells. These biomimetic constructs exhibit aligned architecture, multinucleated and striated myofibers, and a Pax7+cell pool. They contract spontaneously and respond to electrical stimuli with twitch and tetanic contractions. Positive correlation between contractile force and GCaMP6-reported calcium responses enables non-invasive tracking of myobundle function and drug response. During culture, myobundles maintain functional acetylcholine receptors and structurally and functionally mature, evidenced by increased myofiber diameter and improved calcium handling and contractile strength. In response to diversely acting drugs, myobundles undergo dose-dependent hypertrophy or toxic myopathy similar to clinical outcomes. Human myobundles provide an enabling platform for predictive drug and toxicology screening and development of novel therapeutics for muscle-related disorders.

IdMOC per sostituire gli animali in farmacologia, tossicologia, nell’ADMET e nello screening di farmaci anticancro

[Loganathan Gayathri, Dharumadurai Dhanasekaran, and Mohammad A. Akbarsha. Scientific concepts and applications of integrated discrete multiple organ co-culture technology. J Pharmacol Pharmacother. 2015 Apr-Jun; 6(2): 63–70.]

Full Text: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419250/

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Abstract:

“Over several decades, animals have been used as models to investigate the human-specific drug toxicity, but the outcomes are not always reliably extrapolated to the humans in vivo. Appropriate in vitro human-based experimental system that includes in vivo parameters is required for the evaluation of multiple organ interaction, multiple organ/organ-specific toxicity, and metabolism of xenobiotic compounds to avoid the use of animals for toxicity testing. One such versatile in vitro technology in which human primary cells could be used is integrated discrete multiple organ co-culture (IdMOC). IdMOC system adopts wells-within-well concept that facilitates co-culture of cells from different organs in a discrete manner, separately in the respective media in the smaller inner wells which are then interconnected by an overlay of a universal medium in the large containing well. This novel in vitro approach mimics the in vivo situation to a great extent, and employs cells from multiple organs that are physically separated but interconnected by a medium that mimics the systemic circulation and provides for multiple organ interaction. Applications of IdMOC include assessment of multiple organ toxicity, drug distribution, organ-specific toxicity, screening of anticancer drugs, metabolic cytotoxicity, etc.

Conclusioni:

“The IdMOC technology is a simple and new-generation in vitro experimental system which does not require any sophisticated laboratory equipment for the evaluation of distribution, metabolism, and toxicity of a xenobiotic. Co-culture of multiple cell types of the same organ or multiple organs in a physically discrete manner allows the system to interact and helps to predict the multiple endpoints. Use of primary human cells and incorporation of metabolic cytotoxicity to the in vitro system provides an insight to the scientific community that IdMOC is a physiologically relevant model for risk assessment. The embodiment of wells-within-well concept in IdMOC technology has promoted in vitro technique from routine two-dimensional cell culture to mimic, to a great extent, the real in vivoconditions. Thus, IdMOC is an innovative and less time-consuming model that could replace animal testing methods perhaps to comply with the changing regulatory needs. In vitro approach has always been an adoptable technique and readily procures many in vivo key features. Thus, the technique could overcome the uncertainty of animal testing and withstand for a long period to reduce and replace the use of animals in scientific research. However, novel inventions and new methodologies will never stop until the in vitro condition matches or supersedes the in vivo condition. The future of cell culture could be the virtual human-on-chip which may simulate a complete human, but in a simple magnitude. IdMOC has a great potential simulating humans in vivo using in vitro conditions and this technique can be adopted by all researchers who are efficiently carrying out conventional in vitro cell culture in the laboratory.”