Neural progenitor cells represent a cell population, which plays a pivotal role in the development of nervous system during ontogeny, as well as in neural regeneration. Investigation of these processes in humans is greatly restricted due to ethical issues and the limited availability of human neural tissue, especially of the central nervous system (CNS). Most studies on neural development and regeneration are based on animal models, predominantly on primary cultures of high plasticity cells isolated from rodent (1–15) and avian (4, 16, 17) embryos and newborns. Nevertheless, NPCs differentiated from human pluripotent stem cells provide an opportunity to study neural development in human cell-based models.
Neural cells, especially neurons, exhibit a unique polar architecture. Polarization of neural cells is an essential part of neuronal differentiation, thus being fundamental in neural network formation and nerve regeneration. One of the very first episodes of neural polarization is the protrusion of a number of processes from NPCs, which event is followed by the elongation and branching of the evolving neurites. Eventually, one of the projections differentiates into the axon, whereas others develop into dendrites. A fine balance between positive and negative regulatory mechanisms, including both internal and environmental factors, ultimately leads to the establishment and maintenance of the neuronal structure. In the present study, we established an experimental instrumentation, which is suitable for studying the initial step of neural polarization, the neurite outgrowth process in NPCs of human origin. We have differentiated human pluripotent stem cells (ESCs and iPSCs) into neural progenitor cells, using a recent protocol for generation NPCs committed to the hippocampal dentate gyrus lineage (34, 35). Human neural progenitors have a key role in neurogenesis and are potential source for the stem cell-based therapies of neurodegenerative diseases. Moreover, dentate gyrus neural progenitor cells are essential for learning, pattern separation, and spatial memory formation (28); deviations of these cells can cause several disease conditions. Thus, the investigation of neurite polarization mechanism in these cell types can help to improve our strategies on neuro-regeneration and cell-based therapies. Using a transposon-based gene delivery system and cell sorting, we established high-level and stable expression of eGFP in human NPCs. We also demonstrated that eGFP expression did not affected cell growth capacity. High-content microscopy imaging and analysis of these cells allowed us to quantitate the extent and speed of neurite generation, as well as to characterize the developing neurites in term of process number per cell and branching. We found that under basal conditions, human NPCs develop only a small number (1–3) of processes, which are relatively short (30 µm) and have typically no branches (only 1 in 10 neurites).
Development of cell protrusions is complex dynamic process, which is a result of numerous events that involve not only membrane dynamics but also rearrangement of cytoskeletal elements. The structure of neurites is established by stable microtubule bundles and highly dynamic mesh of filamentous actin (F-actin). Critical steps of neurite development include the evolvement of microtubules at the positive ends, their bundling, and the continuous remodeling of actin filaments (40). The ATP-driven molecular motor protein, the non-muscle myosin binds to F-actin and controls the dynamics of actomyosin locomotion, a fundamental biological process, which is connected to numerous cellular and physiological functions including muscle contraction, cell differentiation, migration and polarization (41, 42).
Blebbistatin is a widely used, potent inhibitor of NMII (26, 27), and proved to be a useful tool for studying neuronal differentiation. Treatment of neural cells with blebbistatin results in extensive outgrowth of neurites as a consequence of abolished retrograde actin flow, when the balance of evolvement and retrieval of cell protrusion is shifted to former side. Several studies employed blebbistatin to demonstrate the role of actomyosin contractility using primary neurons from mice (1–5), rats (4–15), chickens (4, 16, 17), or even gastropods, such as Aplysia (18) and Helisoma trivolvis (19). These studies were performed in non-human primary neural cells, and investigated the neurite growth on matured neurons. The role of actomyosin in cannabinoid-induced changes in neuronal morphology was also established by blebbistatin inhibition in primary rat neurons and Neuro2a, murine neuroblastoma cells (10).
Only few studies employed human pluripotent stem cell-derived neural models to investigate the involvement of actomyosin contractility in neural polarization and differentiation. Human ESCs were treated with blebbistatin to demonstrate the role of NMII in topography-induced neuronal maturation (43). Blebbistatin antagonized differentiation of human iPSCs into midbrain dopaminergic neurons induced by mRNAs coding proneural transcription factors, also implying the involvement of NMII (44). A large-scale screening for bioactive small molecules regulating neurite growth identified blebbistatin as a hit compound (108 hits out of 4421) (45). For the high-throughput screen, iCell neurons were used, which are human iPSC-derived, cortical-like neural cultures consisting mostly of GABAergic interneurons. Contrary to the studies above, where differentiated neurons were investigated, we examined the role of NMII in human neural progenitor cells. We found that blebbistatin induces not only elongation of existing neurites but stimulates generation of new projections in human NPCs. Although branch formation of neurites was also stimulated by blebbistatin (∼3-fold), it remained meager at least within the studied time frame (4 hours).
Although blebbistatin is widely used, it has numerous drawbacks when employed in cell biology applications. These include chemical instability, low solubility in water, toxicity to cells, and blue light-induced phototoxicity (29, 32). Blebbistatin also tends to form fluorescent precipitates, which interfere with many cell biology assessments. A-ring modification of blebbistatin results in higher water solubility, but comes at the cost of lower potency for NMII inhibition (46). D-ring-modified blebbistatin analogs, however, such as 3'-hydroxy-blebbistatin, 3'-aminoblebbistatin (47), para-nitroblebbistatin (30), and para-aminoblebbistatin (31), have more favorable properties, like higher water solubility, diminished cytotoxicity, and preserved potency. D-ring-modified BS derivatives with the exception of 3'-hydroxyblebbistatin are non-fluorescent at the spectral range normally used for microscopy or flow cytometry. In the present, study, we investigated the effect of para-nitro- and para-aminoblebbistatin on the growth rate and morphology of neurites in human NPCs. In comparison with blebbistatin, the potency of NBS was similar to that of BS in terms of stimulation of neurite growth, process number, and neurite branching (EC50 values are between 2–5 µM). NBS was also slightly more efficacious. However, AmBS was less potent (EC50 values are around 10 µM) but more efficacious than BS as regards to neurite growth and branching stimulation. The efficacy of AmBS for promoting new protrusion generation was comparable with that of BS. Human NPCs treated with 80 µM AmBS developed 3–4 (3.67 as an average) neurites per cell with the total length of 90–100 µm within 3 hours. As an average, every third neurite developed branch in 3 hours.
NMII is regulated by the phosphorylation of its regulatory light chain on Ser19 and Thr18, which is carried out by a number of kinases, including myosin light chain kinase, Rho-associated, coiled coil-containing kinase, leucine-zipper-interacting protein kinase, citron kinase, Serine/Threonine-protein kinase 21, and myotonic dystrophy kinase-related CDC42-binding kinase (25). An equally important part of NMII regulation is its dephosphorylation by phosphatases, such as the myosin light chain phosphatase. The precise cellular localization of these kinases and phosphatases is crucial for the site-specific regulation of NMII, which is necessary to control filopodia dynamics. ROCK is a key component of various converging signaling pathways of upstream Rho-like GTPases. The regulatory role of MLCK and ROCK in neurite outgrowth has been demonstrated by numerous studies based on animal primary neural cell models (4, 7, 12, 16, 19, 48). A recent report, using wild type and RhoA knockout mice as well as primary hippocampal neuron cultures from these animals, suggested a novel mechanism for Rho/ROCK signaling control of the axonal growth, i.e., ROCK restrains protrusion of microtubules to the leading edge of the growth cone by activating NMII-mediated actin arc formation (3). In concert with the animal-based neural models, we found that ROCK1 inhibition similar to blebbistatin promotes neurite outgrowth in human NPCs. Also, their impact on protrusion initialization and process branching were alike. The stimulatory effect of BS and ROCK1 inhibitor was not additive, confirming that ROCK1 is an upstream regulator of NMII also in a human neural model. These results also suggest that ROCK1 signal is manifested mainly by the NMII activity in the regulation of neurite outgrowth. It is worth noting again that previous studies applying animal-based systems investigated the role of NMII in mature neurons, focusing mainly on the growth cone or the axon initial segment, whereas in the present study, we aim at investigating a precursory event, the initialization of neurite formation in neural progenitors.
JNK signaling pathway is two-faced in neural cells by means of promoting either cell development/regeneration or neuronal death/degeneration depending on the cell type, subcellular localization and cellular condition. A JNK1 knockout animal model demonstrated a pivotal role for JNK1 in neuronal microtubule assembly and stabilization (49). Subsequent studies using spiral ganglion or midbrain dopaminergic neurons from rats confirmed the involvement of all three JNK isoforms, although their differential contributions have also been revealed (39, 50). JNK3 was found to be the most prominent in mediating neurite regeneration and cell survival (39). JNK phosphorylation of downstream effectors, such as the dendrite-specific high-molecular-weight microtubule-associated protein 2 (MAP2) and the microtubule-destabilizing protein SCG10, were shown to contribute to defining dendritic architecture and axodendritic length, respectively (51, 52). Contrary to these studies above, which were performed on mature neurons isolated from rodents, Lu et al. employed mouse embryonic neural stem cells (analog of our NPCs) to examine the involvement of JNK (53). Inhibition of JNK diminished valproic acid-induced neurite outgrowth and neuronal differentiation in these cells. In our hands, the specific JNK inhibitor SP600125 did not block either basal neurite outgrowth, or initiation of processes, or branching in human NPCs. Similarly, blocking the JNK pathway did not affect neurite growth elicited by either NMII or ROCK1 inhibition. Several reasons can be accounted for the different effect of JNK inhibition observed in the previously described cellular models and in our system. Interspecies difference between rodents and humans is one of the plausible explanations. Also, it is known that signaling events greatly dependent on the cell type. NPCs represent an ‘adult’ or more precisely a tissue-specific stem cell population, in which regulatory mechanism can be different from that seen in mature neurons. In addition, as previous studied made it clear, specific cellular localization of kinases in either the RhoA- or the JNK-dependent pathway is crucial for the particular cellular functions (25, 54). It is also noteworthy that we focused only on the initialization, the first 6 hours of neurite generation. Kinetic differences can also be accounted for the conflicting results. Activation of JNK is relatively slow and its kinetics is site-specific in polarized neurons (50). Taken together, we did not see evidence for the involvement of JNK in the regulation of neurite initialization in human NPCs.
Restricted regeneration capability of the central nervous system is determined by both intrinsic and environmental factors. The role of extracellular matrix in neural development and regeneration has long been studied. ECM components in the CNS are produced and secreted by both neurons and glial cells. Some of them, such as laminin and fibronectin, promote neural cell growth and migration, especially in the developing CNS, while others, e.g., chondroitin sulfate proteoglycans, serve as a barrier and prevent axons from growing into improper regions (20). Remodeled ECM constitutes a detrimental environment, imposing a major obstacle for axonal regeneration (21). The effect of these permissive and restrictive ECM components on the neurite outgrowth has been demonstrated in vitro using matured neurons (2, 22–24); however, their impact on neural progenitor cells is poorly studied. In the present study, we investigated the neurite outgrowth of human NPCs on various permissive and restrictive ECMs. We found that laminin is an essential component of ECM to accomplish maximal capacity of basal or NBS-stimulated neurite growth. Matrigel was almost as supportive ECM as the laminin-containing coatings. All studied inhibitory ECM components, including aggrecan, CS, MOG, and CSPG, abolished basal neurite outgrowth. Addition of laminin to these inhibitory mixtures reserved the effect of MOG, but only alleviated the constraining effect of the others. However, blebbistatin derivatives, such as NBS and AmBS, were able to override the ECM inhibition in all cases.
Cell therapies using stem cells or stem cell-derived transplants represent a promising and developing field of regenerative medicine. Several stem cell-based preclinical studies and also a limited number of clinical studies have been performed in connection with various CNS pathologies including neurodegenerative disorders [recently reviewed in (55)]. Rodent models of Alzheimer's disease and Huntington's disease showed marked improvement in behavioral and cognitive deficits after transplantation of human iPSC-derived NPCs (56, 57). Similarly, engraftment of human stem cell-derived NPCs or dopaminergic precursors ameliorated bradykinesia and drug-induced rotation behavior in various animal models of Parkinson's disease (58–60). Moreover, early clinical trials have been launched or yet been forthcoming to explore the safety and efficacy of human iPSC-derived progenitors in Parkinson's disease patients [reviewed in (61)]. Functional recovery was also demonstrated in stroke-damaged rodents subjected to human iPSC-derived NPC transplantation (62, 63). The key issue of these interventions is the functional integration of the transplanted cells. Neuronal polarization starting with protrusions of neurites is a prerequisite for NPCs to integrate. However, intrinsic mechanisms and non-permissive nature of CNS environment for neurite growth greatly impedes this process. Our results demonstrate that targeting NMII can surmount both internal and environmental hindrance of neurite development, offering a new opportunity for improving effectiveness of integration of transplanted cells. Our in vitro data can serve as a base for future in vivo experiments to explore the potential of pharmacological augmentation of cell therapies for various CNS pathologies.