Neuronal cell culture from human embryonic stem cells as in vitro model for neuroprotection

Abstract

In the context of efficacy testing ofpharmacological compounds in animal models, replacement of some of these models with a relevant human in vitro system appears attractive, in particular with regard to large scale screening. Here, we show resultsfrom initial phases of a project, which attempts to explore the outstanding potential of human embryonie stem cell (hESC)based in vitro models with special regard to neuronal stress as a potential replacement of animal models for human neurodegenerative diseases. We show the functionality of neurons derived from hESC precursors by calcium imaging, mitochondrial potential measurements and Western blots and moreover demonstrate that this model reproduces crucial mechanistic aspects observed during ischemia and excitotoxicity that are thought to be at the core of some neurodegenerative diseases. Also, the broader possibilities for refining surrogate molecular information emerging from the detailed analysis of this model are discussed. Zusammenfassung: Stammzellbasierte menschliche in vitro Modelle zum Ersatz der Wirksamkeitsprüfung bei Tieren Im Kontext von Wirksamkeitsstudien von pharmakologischen Formulierungen und Medikamentenkandidaten in Tiermodellen besteht das starke Bedürfnis Ersatzmodelle auf der Basis menschlicher in vitro Systeme zu entwickeln. Wir zeigen hier Ergebnisse aus den frühen Phasen eines Projekts, welches das besondere Potential humaner embryonaler Stammzell-(hESC)-Modelle als entsprechende in vitro Systeme erforschen möchte, mit einem speziellem Fokus auf neuronalen Stress und mit dem Ziel Tiermodelle für humane neurodegenerative Krankheiten zu ersetzen. Wir konnten die Funktionalität von Neuronen, die aus hESC Vorläufern differenziert wurden, durch Calcium Imaging, Potentialmessungen der mitochondrialen Membran und Western Blots zeigen. Wir konnten auch nachweisen, dass das Modell wesentliche mechanistische Aspekte, wie sie während der Ischämie oder unter erregungstoxischen Bedingungen auftreten, gut widerspiegelt. Es wird vermutet, dass diese Prozesse einer Reihe von menschlichen neurodegenerativen Krankheiten zugrunde liegen. Auch weitergehende Möglichkeiten zur Entwicklung besserer und genauerer; auch molekularer Endpunkte, die sich aus der detaillierten Analyse dieser Modelle ergeben könnten, werden diskutiert.

Translating neurobehavioural endpoints of developmental neurotoxicity tests into in vitro assays and readouts

“…A special case of this approach is the investigation of toxicity directly to the ESC (Uibel et al, 2010). More recently, human ESCs have also been used (Stummann et al, 2009; Schrattenholz and Klemm, 2007). The initial phase of differentiation is characterized by particularly strong epigenetic changes (Zimmer et al, 2011a), and it may be susceptible to long-term toxic effects that are manifest only later in life due to epigenetic memory.…”

Systems Biology Approaches and Tools for Analysis of Interactomes and Multi-target Drugs

“…Models like embryonic stem cells and their organ-specific derivatives can be submitted to functional kinetic read-outs by fluorescent techniques, and at the same time being correlated directly to molecular snapshots of biomarker signatures obtained by microarray or differential proteomic techniques (94,95).…”

Synchronization of posttranslational modifications during aging

“…In the upper part 3A, a phase contrast image of a group of neurons is shown; this image remains constant over the course of functional experiments during which these cells are stimulated by, for example, the neurotransmitter glutamate. The same group of neurons shows marked time‐dependent changes due to calcium influx, which can be quantified by Fura‐2 calcium imaging 19 . In the case of excitotoxicity, an excess of calcium leads to mitochondrial apoptosis, which again can be quantified by fluorescent methods.…”

“… Synchronization of functional measurements to differential protein expression on fast time scales: In part A , a phase contrast image of a group of neurons is shown. The same group of neurons is then shown in subsequent calcium images (0 and 30 s after glutamate stimulus), where intracellular calcium concentration differences are quantified by the calcium‐dependent fluorescent dye Fura‐2 19 . In an example in part B , the activation of the mitochondrial permeability transition pore was visualized by efflux of Rhodamin‐123 20 .…”