Overcoming the hurdles for a reproducible generation of human functionally mature reprogrammed neurons

Abstract

The advent of cell reprogramming technologies has widely disclosed the possibility to have direct access to human neurons for experimental and biomedical applications. Human pluripotent stem cells can be instructed in vitro to generate specific neuronal cell types as well as different glial cells. Moreover, new approaches of direct neuronal cell reprogramming can strongly accelerate the generation of different neuronal lineages. However, genetic heterogeneity, reprogramming fidelity, and time in culture of the starting cells can still significantly bias their differentiation efficiency and quality of the neuronal progenies. In addition, reprogrammed human neurons exhibit a very slow pace in gaining a full spectrum of functional properties including physiological levels of membrane excitability, sustained and prolonged action potential firing, mature synaptic currents and synaptic plasticity. This delay poses serious limitations for their significance as biological experimental model and screening platform. We will discuss new approaches of neuronal cell differentiation and reprogramming as well as methods to accelerate the maturation and functional activity of the converted human neurons.

Keywords: Neurons; disease modeling; embryonic stem cells; induced pluripotent stem cells; neuronal activity; synapses.

Figure 1

Morphology and electrophysiological properties of cultured Ngn2 induced neurons. (a) Representative image of MAP2/NeuN double immunostaining of Ngn2-infected iPSC-derived iNs. (b) High-magnification image of a pan-NaV antibody staining correctly localized at the axonal shaft of a Ngn2-iN. In (a) and (b), Hoechst is used as nuclear counterstaining (blue). Scale bar, 50 μm in (a) and 10 μm in (b). (c) Voltage change in response to injection of positive and negative current pulses (+60 and −40 pA, respectively). (d) Spontaneous synaptic activity before and after extracellular perfusion with the AMPA-receptor antagonist NBQX (5 μM). The portion of the trace included in the grey area is magnified below. (A color version of this figure is available in the online journal.)