Test di tossicità cronica usando cellule staminali umane

[Holmgren G, Sjögren AK, Barragan I, Sabirsh A, Sartipy P, Synnergren J, Björquist P, Ingelman-Sundberg M, Andersson TB, Edsbagge J. Long-term chronic toxicity testing using human pluripotent stem cell-derived hepatocytes. Drug Metab Dispos. 2014 Sep;42(9):1401-6.]

Abstract:

”Le cellule staminali umane pluripotenti (HPSC) hanno il potenziale per diventare strumenti importanti per la creazione di nuovi modelli di test dei farmaci in vitro, per esempio, per tossicità ed effetti farmacologici. Il logoramento in fase avanzata nell’industria farmaceutica è in gran parte causato dalla selezione di farmaci candidati che utilizzano modelli preclinici non predittivi che non sono clinicamente rilevanti. Gli attuali modelli epatici in vivo ed in vitro mostrano limiti evidenti, soprattutto per gli studi di epatotossicità cronica. Per queste ragioni, abbiamo valutato il potenziale dell’ utilizzo degli epatociti derivati da cellule staminali umane pluripotenti per l’esposizione a lungo termine ai farmaci tossici. Gli epatociti differenziati sono stati incubati con composti epatotossici fino a 14 giorni, utilizzando un approccio a dosi ripetute. Gli epatociti derivati da cellule staminali umane pluripotenti sono diventati più sensibili ai composti tossici dopo l’esposizione prolungata e, oltre alla citotossicità convenzionale, anche evidenze di fosfolipidosi e steatosi sono state osservate nelle cellule. Questo è, al meglio della nostra conoscenza, il primo rapporto di uno studio di tossicità a lungo termine che ha utilizzato epatociti derivati dalle cellule staminali umane pluripotente, e le osservazioni sostengono l’ulteriore sviluppo e la validazione di modelli di tossicità basati sul loro utilizzo per valutare nuovi farmaci, prodotti chimici e cosmetici.”

Organoidi cerebrali per studiare le malattie neurologiche e lo sviluppo del cervello

[Madeline A Lancaster & Juergen A Knoblich. Generation of cerebral organoids from human pluripotent stem cells. Nature Protocols 9, 2329–2340 (2014).]

Abstract:

Human brain development exhibits several unique aspects, such as increased complexity and expansion of neuronal output, that have proven difficult to study in model organisms. As a result, in vitro approaches to model human brain development and disease are an intense area of research. Here we describe a recently established protocol for generating 3D brain tissue, so-called cerebral organoids, which closely mimics the endogenous developmental program. This method can easily be implemented in a standard tissue culture room and can give rise to developing cerebral cortex, ventral telencephalon, choroid plexus and retinal identities, among others, within 1–2 months. This straightforward protocol can be applied to developmental studies, as well as to the study of a variety of human brain diseases. Furthermore, as organoids can be maintained for more than 1 year in long-term culture, they also have the potential to model later events such as neuronal maturation and survival.

iCRISPR: una nuova tecnica per il genome editing su cellule staminali umane

[González F, Zhu Z, Shi ZD, Lelli K, Verma N, Li QV, Huangfu D. An iCRISPR Platform for Rapid, Multiplexable, and Inducible Genome Editing in Human Pluripotent Stem Cells. Cell Stem Cell. 2014 Aug 7;15(2):215-26.]

Full Text: http://www.cell.com/cell-stem-cell/pdf/S1934-5909(14)00205-7.pdf

Abstract:

Human pluripotent stem cells (hPSCs) offer a unique platform for elucidating the genes and molecular pathways that underlie complex traits and diseases. To realize this promise, methods for rapid and controllable genetic manipulations are urgently needed. By combining two newly developed gene-editing tools, the TALEN and CRISPR/Cas systems, we have developed a genome-engineering platform in hPSCs, which we named iCRISPR. iCRISPR enabled rapid and highly efficient generation of biallelic knockout hPSCs for loss-of-function studies, as well as homozygous knockin hPSCs with specific nucleotide alterations for precise modeling of disease conditions. We further demonstrate efficient one-step generation of double- and triple-gene knockout hPSC lines, as well as stage-specific inducible gene knockout during hPSC differentiation. Thus the iCRISPR platform is uniquely suited for dissection of complex genetic interactions and pleiotropic gene functions in human disease studies and has the potential to support high-throughput genetic analysis in hPSCs.

Comunicazione cervello-cervello negli esseri umani usando tecniche non-invasive

[Grau C, Ginhoux R, Riera A, Nguyen TL, Chauvat H, et al. (2014) Conscious Brain-to-Brain Communication in Humans Using Non-Invasive Technologies. PLoS ONE 9(8): e105225.]

Full Text: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0105225

Abstract:

Human sensory and motor systems provide the natural means for the exchange of information between individuals, and, hence, the basis for human civilization. The recent development of brain-computer interfaces (BCI) has provided an important element for the creation of brain-to-brain communication systems, and precise brain stimulation techniques are now available for the realization of non-invasive computer-brain interfaces (CBI). These technologies, BCI and CBI, can be combined to realize the vision of non-invasive, computer-mediated brain-to-brain (B2B) communication between subjects (hyperinteraction). Here we demonstrate the conscious transmission of information between human brains through the intact scalp and without intervention of motor or peripheral sensory systems. Pseudo-random binary streams encoding words were transmitted between the minds of emitter and receiver subjects separated by great distances, representing the realization of the first human brain-to-brain interface. In a series of experiments, we established internet-mediated B2B communication by combining a BCI based on voluntary motor imagery-controlled electroencephalographic (EEG) changes with a CBI inducing the conscious perception of phosphenes (light flashes) through neuronavigated, robotized transcranial magnetic stimulation (TMS), with special care taken to block sensory (tactile, visual or auditory) cues. Our results provide a critical proof-of-principle demonstration for the development of conscious B2B communication technologies. More fully developed, related implementations will open new research venues in cognitive, social and clinical neuroscience and the scientific study of consciousness. We envision that hyperinteraction technologies will eventually have a profound impact on the social structure of our civilization and raise important ethical issues.

Modello microfluidico di sistema circolatorio umano

[Tourovskaia A, Fauver M, Kramer G, Simonson S, Neumann T. Tissue-engineered microenvironment systems for modeling human vasculature. Exp Biol Med (Maywood). 2014 Sep;239(9):1264-71.]

Abstract:

The high attrition rate of drug candidates late in the development process has led to an increasing demand for test assays that predict clinical outcome better than conventional 2D cell culture systems and animal models. Government agencies, the military, and the pharmaceutical industry have started initiatives for the development of novel in-vitro systems that recapitulate functional units of human tissues and organs. There is growing evidence that 3D cell arrangement, co-culture of different cell types, and physico-chemical cues lead to improved predictive power. A key element of all tissue microenvironments is the vasculature. Beyond transporting blood the microvasculature assumes important organ-specific functions. It is also involved in pathologic conditions, such as inflammation, tumor growth, metastasis, and degenerative diseases. To provide a tool for modeling this important feature of human tissue microenvironments, we developed a microfluidic chip for creating tissue-engineered microenvironment systems (TEMS) composed of tubular cell structures. Our chip design encompasses a small chamber that is filled with an extracellular matrix (ECM) surrounding one or more tubular channels. Endothelial cells (ECs) seeded into the channels adhere to the ECM walls and grow into perfusable tubular tissue structures that are fluidically connected to upstream and downstream fluid channels in the chip. Using these chips we created models of angiogenesis, the blood-brain barrier (BBB), and tumor-cell extravasation. Our angiogenesis model recapitulates true angiogenesis, in which sprouting occurs from a “parent” vessel in response to a gradient of growth factors. Our BBB model is composed of a microvessel generated from brain-specific ECs within an ECM populated with astrocytes and pericytes. Our tumor-cell extravasation model can be utilized to visualize and measure tumor-cell migration through vessel walls into the surrounding matrix. The described technology can be used to create TEMS that recapitulate structural, functional, and physico-chemical elements of vascularized human tissue microenvironments in vitro.

Modelli organotipici per sostituire gli animali con sistemi più rilevanti

[BéruBé K, Gibson C, Job C, Prytherch Z. Human lung tissue engineering: a critical tool for safer medicines. Cell Tissue Bank. 2011 Feb;12(1):11-3.]

Abstract:

In the field of human tissue-engineering, there has been a strong focus on the clinical aspects of the technology, i.e. repair, replace and enhance a given tissue/organ. However, much wider applications for tissue engineering (TE) exist outside of the clinic that are often not recognised, and include engineering more relevant models than animals in basic research and safety testing. Traditionally, research is initially conducted on animals or cell lines, both of which have their limitations. With regard to cell lines, they are usually transformed to enable indefinite proliferation. These immortalised cell lines provide the researcher with an almost limitless source of material. However, the pertinence of the data produced is now under scrutiny, with the suggestion that some historical cell lines may not be the cell type originally reported. By engineering normal, biomimetic (i.e. life-mimicking), human tissues with defined physiology (i.e. human tissue equivalents), the complex 3-dimensional (3-D) tissue/organ physiology is captured in vitro, providing the opportunity to directly replace the use of animals in research/testing with more relevant systems. Therefore, it is imperative that testing strategies using organotypic models are developed that can address the limitations of current animal and cellular models and thus improve drug development, enabling faster delivery of drugs which are safer, more effective and have fewer side effects in humans.

 

Organoidi di cellule staminali come modello dello sviluppo umano per studi di organogenesi e teratogenesi

[Ader M, Tanaka EM. Modeling human development in 3D culture. Curr Opin Cell Biol. 2014 Jul 14;31C:23-28.]

Abstract:

Recently human embryonic stem cell research has taken on a new dimension – the third dimension. Capitalizing on increasing knowledge on directing pluripotent cells along different lineages, combined with ECM supported three-dimensional culture conditions, it has become possible to generate highly organized tissues of the central nervous system, gut, liver and kidney. Each system has been used to study different aspects of organogenesis and function including physical forces underlying optic cup morphogenesis, the function of disease related genes in progenitor cell control, as well as interaction of the generated tissues with host tissue upon transplantation. Pluripotent stem cell derived organoids represent powerful systems for the study of how cells self-organize to generate tissues with a given shape, pattern and form.

 

Quasi-Vivo: studi di tossicità e interazioni tra organi e sistemi senza usare animali

[Sbrana T, Ahluwalia A. Engineering Quasi-Vivo in vitro organ models. Adv Exp Med Biol. 2012;745:138-53.]

Abstract:

Cell culture is the workhorse of biologists, toxicologists, tissue engineers and a whole host of research fields in both academia and industry. Having explored individual molecular mechanisms inside cells for decades using traditional cell culture techniques, researchers have only just begun to appreciate that the intricate interconnectivity between cells and cellular networks as well as with the external environment is far more important to cellular orchestration than are single molecular events inside the cell. For example many questions regarding cell, tissue, organ and system response to drugs, environmental toxins, stress and nutrients cannot possibly be answered by concentrating on the minutiae of what goes on in the deepest recesses of single cells. New models are required to investigate cellular cross-talk between different cell types and to construct complex in-vitro models to properly study tissue, organ and system interaction without resorting to animal experiments. This chapter describes how tissue and organ models can be developed using the Quasi-Vivo system and discusses how they may be used in drug toxicity studies.

IdMOC ed epatociti in vitro per valutare l’ADMET, il metabolismo, le interazioni farmaco-farmaco e la tossicità nello sviluppo dei farmaci

[Li AP. In vitro human hepatocyte-based experimental systems for the evaluation of human drug metabolism, drug-drug interactions, and drug toxicity in drug development. Curr Top Med Chem. 2014;14(11):1325-38.]

Abstract:

Species difference in drug metabolism and drug toxicity is a well-established phenomenon. As a result, the classical paradigm of preclinical testing of drug candidates in animals may not be appropriate. One preclinical approach to evaluate human drug properties, especially ADMET (absorption, disposition, metabolism, elimination, and toxicity) properties, is to apply in vitro experimental systems with relevant human properties. The latest advances include the use of human hepatocytes to evaluate hepatic uptake, metabolism, efflux and toxicity. Successful cryopreservation of human hepatocytes to retain high viability, metabolic capacity, as well as the ability to be cultured allow routine application of this relevant experimental system. This review summarizes the latest findings on human hepatocytes isolation, cryopreservation, culturing, as well as application in the evaluation of metabolic stability, metabolite profiling, hepatic uptake and efflux, metabolic drug-drug interactions, and drug toxicity. The use of hepatocyte to evaluate the role of metabolism in drug toxicity represents a major advance in drug toxicity evaluation. The use of the novel integrated discrete multiple organ coculture (IdMOC) system allows the evaluation of the role of hepatic metabolism on nonhepatic toxicity.

Cellule staminali come modelli di malattie neurodegenerative e per il drug discovery

[Cao L, Tan L, Jiang T, Zhu XC, Yu JT. Induced Pluripotent Stem Cells for Disease Modeling and Drug Discovery in Neurodegenerative Diseases.  Mol Neurobiol. 2014 Aug 23. [Epub ahead of print]]

Abstract:

Although most neurodegenerative diseases have been closely related to aberrant accumulation of aggregation-prone proteins in neurons, understanding their pathogenesis remains incomplete, and there is no treatment to delay the onset or slow the progression of many neurodegenerative diseases. The availability of induced pluripotent stem cells (iPSCs) in recapitulating the phenotypes of several late-onset neurodegenerative diseases marks the new era in in vitro modeling. The iPSC collection represents a unique and well-characterized resource to elucidate disease mechanisms in these diseases and provides a novel human stem cell platform for screening new candidate therapeutics. Modeling human diseases using iPSCs has created novel opportunities for both mechanistic studies as well as for the discovery of new disease therapies. In this review, we introduce iPSC-based disease modeling in neurodegenerative diseases, such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and amyotrophic lateral sclerosis. In addition, we discuss the implementation of iPSCs in drug discovery associated with some new techniques.