Pharmacological Modulation of Neurite Outgrowth in Human Neural Progenitor Cells by Inhibiting Non-muscle Myosin II

Abstract

Studies on neural development and neuronal regeneration after injury are mainly based on animal models. The establishment of pluripotent stem cell (PSC) technology, however, opened new perspectives for better understanding these processes in human models by providing unlimited cell source for hard-to-obtain human tissues. Here, we aimed at identifying the molecular factors that confine and modulate an early step of neural regeneration, the formation of neurites in human neural progenitor cells (NPCs). Enhanced green fluorescent protein (eGFP) was stably expressed in NPCs differentiated from human embryonic and induced PSC lines, and the neurite outgrowth was investigated under normal and injury-related conditions using a high-content screening system. We found that inhibitors of the non-muscle myosin II (NMII), blebbistatin and its novel, non-toxic derivatives, initiated extensive neurite outgrowth in human NPCs. The extracellular matrix components strongly influenced the rate of neurite formation but NMII inhibitors were able to override the inhibitory effect of a restrictive environment. Non-additive stimulatory effect on neurite generation was also detected by the inhibition of Rho-associated, coiled-coil-containing protein kinase 1 (ROCK1), the upstream regulator of NMII. In contrast, inhibition of c-Jun N-terminal kinases (JNKs) had only a negligible effect, suggesting that the ROCK1 signal is dominantly manifested by actomyosin activity. In addition to providing a reliable cell-based in vitro model for identifying intrinsic mechanisms and environmental factors responsible for impeded axonal regeneration in humans, our results demonstrate that NMII and ROCK1 are important pharmacological targets for the augmentation of neural regeneration at the progenitor level. These studies may open novel perspectives for development of more effective pharmacological treatments and cell therapies for various neurodegenerative disorders.

Microglia modulate proliferation, neurite generation and differentiation of human neural progenitor cells

“…Total neurite outgrowth at different time points was determined on the basis of green fluorescence and morphological parameters, whereas the corresponding cell numbers of GFP-NPCs were quantified based on the nuclear staining (Figure 2A). We found that untreated GFP-NPCs 48 h after plating gradually develop protrusions at a rate of 7.09 ± 2.03 mm/cell/48 h (~80% increase within the following 48 h), which is consistent with our previous findings on neurite growth rate of unstimulated GFP-NPCs grown on pOrn/Lam coated surface (Lilienberg et al, 2021). NPCs under the applied conditions exhibit a moderate proliferation rate (cell number increased by ~20% per day, the equivalent of a doubling time of 4 days).…”

“…The neural progenitor cell lines used in this study have previously been generated and characterized in detail ( Yu et al, 2014 ; Vofely et al, 2018 ; Lilienberg et al, 2021 ). These cells express no pluripotency markers, such as Nanog and Oct4; but the typical neural progenitor markers, such as Nestin and SOX2; as well as the neuronal markers, such as NeuroD1, FOXG1, and PAX6 ( Yu et al, 2014 ; Vofely et al, 2018 ).…”

“…Human neural progenitor cells were previously generated from an induced pluripotent stem cell line (62F, obtained from Fred H. Gage, Salk Institute, United States) using an established directed differentiation protocol ( Yu et al, 2014 ) slightly modified by ( Lilienberg et al, 2021 ). Also in a previous work, the green fluorescent protein (GFP) was stably expressed in these NPCs using a Sleeping Beauty transposon-based gene delivery system ( Kolacsek et al, 2014 ), and the cells expressing GFP at high levels were sorted out by flow cytometry, establishing the GFP-NPC line ( Lilienberg et al, 2021 ).…”

“…536/40 nm) with a 10× Nikon objective (Plan Fluor, NA = 0.3). Total outgrowth and cell number was quantified from the green and blue (GFP and DCV) fluorescence images, respectively ( Lilienberg et al, 2021 ). Average neurite length per cell was calculated by dividing the total neurite length by the cell number.…”

“…Images were acquired every 15 min for 4 h using a high content screening microscope. The rate of neurite outgrowth was determined as described previously ( Lilienberg et al, 2021 ).…”