Ion channels and ionotropic receptors in human embryonic stem cell derived neural progenitors

Abstract

Human neural progenitor cells differentiated from human embryonic stem cells offer a potential cell source for studying neurodegenerative diseases and for drug screening assays. Previously, we demonstrated that human neural progenitors could be maintained in a proliferative state with the addition of leukemia inhibitory factor and basic fibroblast growth factor. Here we demonstrate that 96 hours after removal of basic fibroblast growth factor the neural progenitor cell culture was significantly altered and cell replication halted. 14 days after the removal of basic fibroblast growth factor, most cells expressed MAP2 and TUJ1, markers characterizing a post-mitotic neuronal phenotype as well as neural developmental markers Cdh2 and Gbx2. Real-time PCR was performed to determine the ionotrophic receptor subunit expression profile. Differentiated neural progenitors express subunits of glutamatergic, GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium and calcium channel subunits were also expressed. Functionally, virtually all the NP cells tested under whole-cell voltage clamp exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited tetrodotoxin-sensitive, voltage-dependent sodium channel current. Action potentials could also be elicited by current injection under whole-cell current clamp in a minority of cells. These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and has the capability to produce excitable cells that can generate action potentials, a landmark characteristic of a neuronal phenotype. This is the first report of an efficient and simple means of generating human neuronal cells for ionotrophic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.

Neurotrophic Effects of Leukemia Inhibitory Factor on Neural Cells Derived from Human Embryonic Stem Cells

“…4) imply that a greater number of cells reach a more advanced differentiated stage and express neuronal markers, even though an equal percentage exited the progenitor state. Given that these NP cells do not differentiate to glial cells under the conditions used in this study [70], present results implied a positive role of LIF for increasing the yield of pan-neuronal cells during in vitro differentiation.…”

“…This implies that a certain proportion of cells counted as NP cells could be early neurons. HuC/D is a RNA binding protein expressed in the cell bodies of early postmitotic neurons [66][67][68][69][70][71][72][73][74][75][76][77][78], and MAP2 marks dendrites of differentiated neurons [69] (Fig. 4).…”

Plasticity of Calcium Signaling Cascades in Human Embryonic Stem Cell-Derived Neural Precursors

“…In neural progenitors derived from hESCs after 30 days in culture, Malmersjo et al observed an increase in [Ca 2 + ] i evoked by 50 mM K + , but they did not specify which type of VOCC was activated [22]. Using reverse transcript (RT)-polymerase chain reaction (PCR) studies, others have shown the upregulation of VOCC expression in differentiated hESCs [23]. L-type channels are also expressed in NPs derived from human [25] and mouse ESCs [24].…”

“…To the best of our knowledge there is only 1 study by Young et al [23] that has demonstrated the presence of purinergic receptors in hESC-derived neural progenitors. Though the authors did not perform functional studies on P2 receptors, they showed by RT-PCR that in the hESC line WA09, the P2X 4 subunits are upregulated in hNPs but downregulated in differentiated hNPs, while P2X 7 was not expressed at any stage.…”

“…To our knowledge there are only a few reports available on mouse ESCs [20,21] and on hESCs differentiated toward neurones [22][23][24][25]. Therefore, we undertook the present study to determine whether the functional properties of NPs change during long-term maintenance in culture.…”