High-Throughput Screening-Compatible Single-Step Protocol to Differentiate Embryonic Stem Cells in Neurons

Abstract

Biotechnologies such as high-throughput screening (HTS) enable evaluation of large compound libraries for their biological activity and toxic properties. In the field of drug development, embryonic stem (ES) cells have been instrumental in HTS for testing the effect of new compounds. We report an innovative method in one step to differentiate ES cells in neurons and glial cells. The four different neuronal subtypes, gamma-aminobutyric acid (GABA)-ergic, dopaminergic, serotonergic, and motor neurons, are formed in culture. This protocol is adaptable to small wells and is highly reproducible, as indicated by the Z-factor value. Moreover, by using either leukemia inhibitory factor (LIF) or recombinant Cripto protein in our culture conditions, we provide evidence that this protocol is suitable for testing the effect of different molecules on neuronal differentiation of ES cells. Finally, thanks to the simplicity in carrying out the experiment, this method provides the possibility of following the morphological evolution of the in vitro differentiating neuronal cells by timelapse videomicroscopy. Our experimental system provides a powerful tool for testing the effect of different substances on survival and/or differentiation of neuronal and glial cells in an HTS-based approach. Furthermore, using genetically modified ES cells, it would be possible to screen for drugs that have a therapeutic effect on specific neuronal pathologies or to follow, by time-lapse videomicroscopy, their ability to in vitro differentiate.

Ciliopathy proteins regulate paracrine signaling by modulating proteasomal degradation of mediators

“…We turned to an established cilium-dependent paracrine signaling pathway, Hedgehog signaling (Hh) (2), and developed an in vitro neural differentiation model, in which the embryonic WT murine stem cells (WTESs) and Ofd1-deficient murine embryonic stem cells (KOESs) were cultured to generate neural progenitors (Supplemental Figure 2), a model that recapitulates the multi-step process of neural development in embryos (33). KOESs are male murine cells containing a gene trap mutation in Ofd1.…”

Early tissue transglutaminase–mediated response underlies K562(S)-cell gliadin-dependent agglutination

“…After 48 h, cells were incubated with 40 µg/ml of p31-43, and the dish was placed in a time-lapse video microscopy workstation (35). The system was equipped with an inverted optical microscope (Zeiss, Obercoken, Germany) enclosed in a plexiglass cage where a prewarmed air stream at 37 °C mixed with CO 2 was circulated to mimic the conditions of a bench incubator for cell cultures.…”

Embryonic Stem Cells: from Blastocyst to in vitro Differentiation

“…In the second method, ES cells are cultured directly on stromal cells, and differentiation takes place in contact with these cells (Nakano et al, 1994). The third protocol involves differentiating ES cells in a monolayer on extracellular matrix proteins (Nishikawa et al, 1998) or in presence of specific differentiation medium (Takahashi et al, 2003;Fico et al, 2008).…”

“…The various approaches include (1) treatment of serum-stimulated EBs with retinoic acid (Bain et al, 1995), (2) sequential culture of EBs in serum followed by serum-free medium (Okabe et al, 1996), (3) differentiation of ES cells as a monolayer in serum-free medium (Tropepe et al, 2001;Ying et al, 2003;Fico et al, 2008), and (4) differentiation of ES cells directly on stromal cells in the absence of serum (Kawasaki et al, 2000;Barberi et al, 2003). As with the mesoderm and endoderm lineages, development of the ectoderm lineages in the ES differentiation cultures appears to recapitulate their development in the early embryo (Barberi et al, 2003).…”

“…In 2008, Fico et al established a one-step protocol that allowed differenziation of mouse ES cells into a highly enriched population of neuronal cells, simply by cul t u r i n g t h e m o n g e l a t i n -c o a t e d d i s h e s i n a chemically defined serum-free medium. This differentiation method is able to generate a wide range of neural subtypes and glial cells from mouse ES cells (Fico et al, 2008). The ability to generate different types of neurons from ES cells has dramatically raised the interest in repair of nervous system disorders by cell replacement therapy.…”