In Vitro Generation of Posterior Motor Neurons from Human Pluripotent Stem Cells
The ability to generate spinal cord motor neurons from human pluripotent stem cells (hPSCs) is of great use for modelling motor neuron–based diseases and cell-replacement therapies. A key step in the design of hPSC differentiation strategies aiming to produce motor neurons involves induction of the appropriate anteroposterior (A-P) axial identity, an important factor influencing motor neuron subtype specification, functionality, and disease vulnerability. Most current protocols for induction of motor neurons from hPSCs produce predominantly cells of a mixed hindbrain/cervical axial identity marked by expression of Hox paralogous group (PG) members 1-5, but are inefficient in generating high numbers of more posterior thoracic/lumbosacral Hox PG(8-13)+ spinal cord motor neurons. Here, we describe a protocol for efficient generation of thoracic spinal cord cells and motor neurons from hPSCs. This step-wise protocol relies on the initial generation of a neuromesodermal-potent axial progenitor population, which is differentiated first to produce posterior ventral spinal cord progenitors and subsequently to produce posterior motor neurons exhibiting a predominantly thoracic axial identity. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.
Generating Enteric Nervous System Progenitors from Human Pluripotent Stem Cells
The intrinsic innervation of the gastrointestinal (GI) tract is comprised of enteric neurons and glia, which are buried within the wall of the bowel and organized into two concentric plexuses that run along the length of the gut forming the enteric nervous system (ENS). The ENS regulates vital GI functions including gut motility, blood flow, fluid secretion, and absorption and thus maintains gut homeostasis. During vertebrate development it originates predominantly from the vagal neural crest (NC), a multipotent cell population that emerges from the caudal hindbrain region, migrates to and within the gut to ultimately generate neurons and glia in response to gut-derived signals. Loss of GI innervation due to congenital or acquired defects in ENS development causes enteric neuropathies which lack curative treatment. Human pluripotent stem cells (hPSCs) offer a promising in vitro source of enteric neurons for modeling human ENS development and pathology and potential use in cell therapy applications. Here we describe in detail a differentiation strategy for the derivation of enteric neural progenitors and neurons from hPSCs through a vagal NC intermediate. Using a combination of instructive signals and retinoic acid in a dose/time dependent manner, vagal NC cells commit into the ENS lineage and develop into enteric neurons and glia upon culture in neurotrophic media. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC.
Electrical monitoring approaches in 3-dimensional cell culture systems: Toward label-free, high spatiotemporal resolution, and high-content data collection in vitro
3-dimensional (3D) cell cultures better mimic natural environment of cells than 2-dimensional (2D) cell cultures to obtain in vivo like inter and intracellular responses. However, third dimension brings complexity to cell culture. Therefore, high-resolution/high-content screening in 3D is one of the most important challenges with this type of cell cultures. Although optical monitoring techniques, well-established in 2D area, are enhanced to monitor 3D cell cultures, they are generally endpoint, static, time inefficient, and labor intensive. Alternatively, electrical sensing can become a solution to achieve dynamic, real-time, and label-free monitoring of cells in both 2D and 3D cell cultures. Developments in electrical monitoring of cell culture have led to novel approaches, proposed by adapting fundamentals of 2D electrical techniques to 3D to obtain high spatiotemporal systems. In this review, we classified these approaches into five main groups: (i) 3D impedance measurement approach (ii) electrical impedance tomography, (iii) 3D microelectrode array approach, (iv) 3D nanoelectronics scaffold approach, and (v) microphysiometry. We also defined the challenges in the adaptation of electrical monitoring techniques to 3D cultures and explained possible solutions in terms of specific applications and technical point of views, including methods particular to our group. In conclusion, 3D electrical monitoring in cell cultures is considerably challenging but highly accelerated recently by significant advances of microfabrication technology, bioengineering, and material science. Novel approaches reviewed here have a lot of potential and offer opportunities for further developments to find solutions, fit to serve the (bio)medical needs.
Analyzing Developing Brain-On-Chip Cultures with the CALIMA Calcium Imaging Tool
Brain-on-chip (BoC) models are tools for reproducing the native microenvironment of neurons, in order to study the (patho)physiology and drug-response of the brain. Recent developmentsin BoC techniques focus on steering neurons in their activity via microfabrication and via computer-steered feedback mechanisms. These cultures are often studied through calcium imaging (CI), a method for visualizing the cellular activity through infusing cells with a fluorescent dye. CAlciumImagingAnalyser 2.0 (CALIMA 2.0) is an updated version of a software tool that detects and analyzes fluorescent signals and correlates cellular activity to identify possible network formation in BoC cultures. Using three previous published data sets, it was demonstrated that CALIMA 2.0 can analyze large data sets of CI-data and interpret cell activity to help study the activity and maturity of BoC cultures. Last, an analysis of the processing speed shows that CALIMA 2.0 is sufficiently fast to process data sets with an acquisition rate up to 5 Hz in real-time on a medium-performance computer.
Read more: https://doi.org/10.3390/mi12040412
Defining the signalling determinants of a posterior ventral spinal
cord identity in human neuromesodermal progenitor derivatives
The anteroposterior axial identity of motor neurons (MNs) determines their functionality and vulnerability to neurodegeneration. Thus, it is a crucial parameter in the design of strategies aiming to produce MNs from human pluripotent stem cells (hPSCs) for regenerative medicine/disease modelling applications. However, the in vitro generation of posterior MNs corresponding to the thoracic/lumbosacral spinal cord has been challenging. Although the induction of cells resembling neuromesodermal progenitors (NMPs), the bona fide precursors of the spinal cord, offers a promising solution, the progressive specification of posterior MNs from these cells is not well defined. Here, we determine the signals guiding the transition of human NMP-like cells toward thoracic ventral spinal cord neurectoderm. We show that combined WNT-FGF activities drive a posterior dorsal pre-/early neural state, whereas suppression of TGFβ-BMP signalling pathways promotes a ventral identity and neural commitment. Based on these results, we define an optimised protocol for the generation of thoracic MNs that can efficiently integrate within the neural tube of chick embryos. We expect that our findings will facilitate the comparison of hPSC-derived spinal cord cells of distinct axial identities.
Stiff-to-Soft Transition from Glass to 3D Hydrogel Substrates in Neuronal Cell Culture
Over the past decade, hydrogels have shown great potential for mimicking three- dimensional (3D) brain architectures in vitro due to their biocompatibility, biodegradability, and wide range of tunable mechanical properties. To better comprehend in vitro human brain models and the mechanotransduction processes, we generated a 3D hydrogel model by casting photo-polymerized gelatin methacryloyl (GelMA) in comparison to poly (ethylene glycol) diacrylate (PEGDA) atop of SH-SY5Y neuroblastoma cells seeded with 150,000 cells/cm2 according to our previous experience in a microliter-sized polydimethylsiloxane (PDMS) ring serving for confinement. 3D SH-SY5Y neuroblastoma cells in GelMA demonstrated an elongated, branched, and spreading morphology resembling neurons, while the cell survival in cast PEGDA was not supported. Confocal z-stack microscopy confirmed our hypothesis that stiff-to-soft material transitions promoted neuronal migration into the third dimension. Unfortunately, large cell aggregates were also observed. A subsequent cell seeding density study revealed a seeding cell density above 10,000 cells/cm2 started the formation of cell aggregates, and below 1500 cells/cm2 cells still appeared as single cells on day 6. These results allowed us to conclude that the optimum cell seeding density might be between 1500 and 5000 cells/cm2. This type of hydrogel construct is suitable to design a more advanced layered mechanotransduction model toward 3D microfluidic brain-on-a-chip applications.
Read more: https://www.mdpi.com/2072-666X/12/2/165
Three-Dimensional Fine Structure of Nanometer-Scale Nafion Thin
Nafion is a widely used polymer membrane in various applications ranging from advanced energy solutions to sensing of biomolecules. Despite the intensive research carried out over the years to reveal and understand the fine structure of Nafion, its structural features, especially as nanometer-scale films, are not unambiguously known. In this paper, we use room temperature scanning transmission electron microscopy (STEM) tomography complemented by glancing incidence small-angle X-ray scattering (GISAXS) and TEM at low temperatures to reveal the fine structure of thin (10−100 nm) unannealed Nafion films. The results from the detailed three-dimensional reconstructions obtained show that (i) the phase fractions of the hydrophobic and hydrophilic parts of the polymer are somewhat thickness-dependent, changing from 0.65/0.35 to about 0.7/0.3 when moving from 100 to 10 nm thick films; (ii) the channel diameters show a range of values from 3 to 6 nm in all the films independent of their thickness; (iii) the average distances between the hydrophilic channels inside the film have distributions centered around 12 nm (in 10 nm films), 15 nm (in 30 nm films), and 7 nm (in 100 nm films); (iv) in the thickest films, the hydrophilic channels exhibit higher interconnectivity and some of the channels appear to end within the Nafion film instead of going through the films; and (v) there are some confinement effects caused by the hydrophilic SiO2 surface in the case of 10 and 30 nm thick films shown by the tendency of the hydrophilic channels to move horizontally near the substrate. Furthermore, a stable room temperature STEM tomography imaging method for Nafion films and a sample preparation method that preserves the characteristics of the hydrated morphology of Nafion in the dry state are demonstrated. These results provide a deeper understanding of the fine structure of Nafion thin films and provide a better means to characterize and understand their properties in different applications.
Read more: https://dx.doi.org/10.1021/acsapm.0c01318
Trends in Carbon, Oxygen, and Nitrogen Core in the X-ray Absorption Spectroscopy of Carbon Nanomaterials: A Guide for the Perplexed
Successful deployment of carbon nanomaterials in many applications, such as sensing, energy storage, and catalysis, relies on the selection, synthesis, and tailoring of the surface properties. Predictive analysis of the behavior is difficult without detailed knowledge of the differences between various carbon nanomaterials and their surface functionalization, thus leaving the selection process to traditional trial-and-error work. The present characterization fills this knowledge gap for carbon nanomaterial surface properties with respect to chemical states and functionalization. We present an overview of the chemical trends that can be extracted from soft X-ray absorption spectroscopy (XAS) spectra on an extended set of nonideal carbon nanomaterials as a function of sp2 bonded carbon and bond ordering. In particular, the surface chemical state, the presence of long-range order in the carbon matrix, and a qualitative estimation of the amount of oxygen and nitrogen and their respective functional group formation on the material surface, together with the detailed material fabrication parameters, are reported. The results expand our understanding of carbon nanomaterial functionalization, which can support material selection in practice, provided that the specifications of the application are known.
Gut innervation and enteric nervous system development: a spatial, temporal and molecular tour de force
During embryonic development, the gut is innervated by intrinsic (enteric) and extrinsic nerves. Focusing on mammalian ENS development, in this Review we highlight how important the different compartments of this innervation are to assure proper gut function. We specifically address the three-dimensional architecture of the innervation, paying special attention to the differences in development along the longitudinal and circumferential axes of the gut. We review recent information about the formation of both intrinsic innervation, which is fairly well-known, as well as the establishment of the extrinsic innervation, which, despite its importance in gut-brain signaling, has received much less attention. We further discuss how external microbial and nutritional cues or neuroimmune interactions may influence development of gut innervation. Finally, we provide summary tables, describing the location and function of several well-known molecules, along with some newer factors that have more recently been implicated in the development of gut innervation.
Gaining Micropattern Fidelity in an NOA81 Microsieve Laser Ablation Process
We studied the micropattern fidelity of a Norland Optical Adhesive 81 (NOA81) microsieve made by soft-lithography and laser micromachining. Ablation opens replicated cavities, resulting in three-dimensional (3D) micropores. We previously demonstrated that microsieves can capture cells by passive pumping. Flow, capture yield, and cell survival depend on the control of the micropore geometry and must yield high reproducibility within the device and from device to device. We investigated the NOA81 film thickness, the laser pulse repetition rate, the number of pulses, and the beam focusing distance. For NOA81 films spin-coated between 600 and 1200 rpm, the pulse number controls the breaching of films to form the pore’s aperture and dominates the process. Pulse repetition rates between 50 and 200 Hz had no observable influence. We also explored laser focal plane to substrate distance to find the most effective ablation conditions. Scanning electron micrographs (SEM) of focused ion beam (FIB)-cut cross sections of the NOA81 micropores and inverted micropore copies in polydimethylsiloxane (PDMS) show a smooth surface topology with minimal debris. Our studies reveal that the combined process allows for a 3D micropore quality from device to device with a large enough process window for biological studies.
Integrated, automated maintenance, expansion and differentiation of 2D and 3D patient-derived cellular models for high throughput drug screening
Patient-derived cellular models become an increasingly powerful tool to model human diseases for precision medicine approaches. The identification of robust cellular disease phenotypes in these models paved the way towards high throughput screenings (HTS) including the implementation of laboratory advanced automation. However, maintenance and expansion of cells for HTS remains largely manual work. Here, we describe an integrated, complex automated platform for HTS in a translational research setting also designed for maintenance and expansion of different cell types. The comprehensive design allows automation of all cultivation steps and is flexible for development of methods for variable cell types. We demonstrate protocols for controlled cell seeding, splitting and expansion of human fibroblasts, induced pluripotent stem cells (iPSC), and neural progenitor cells (NPC) that allow for subsequent differentiation into different cell types and image-based multiparametric screening. Furthermore, we provide automated protocols for neuronal differentiation of NPC in 2D culture and 3D midbrain organoids for HTS. The flexibility of this multitask platform makes it an ideal solution for translational research settings involving experiments on different patient-derived cellular models for precision medicine.
Nanogrooves for 2D and 3D Microenvironments of SH-SY5Y Cultures in Brain-on-Chip Technology
Brain-on-chip (BOC) technology such as nanogrooves and microtunnel structures can advance in vitro neuronal models by providing a platform with better means to maintain, manipulate and analyze neuronal cell cultures. Specifically, nanogrooves have been shown to influence neuronal differentiation, notably the neurite length and neurite direction. Here, we have drawn new results from our experiments using both 2D and 3D neuronal cell culture implementing both flat and nanogrooved substrates. These are used to show a comparison between the number of cells and neurite length as a first indicator for valuable insights into baseline values and expectations that can be generated from these experiments toward design optimization and predictive value of the technology in our BOC toolbox. Also, as a new step toward neuronal cell models with multiple compartmentalized neuronal cell type regions, we fabricated microtunnel devices bonded to both flat and nanogrooved substrates to assess their compatibility with neuronal cell culture. Our results show that with the current experimental protocols using SH-SY5Y cells, we can expect 200 – 400 cells with a total neurite length of approximately 4,000–5,000 μm per 1 mm2 within our BOC devices, with a lower total neurite length for 3D neuronal cell cultures on flat substrates only. There is a statistically significant difference in total neurite length between 2D cell culture on nanogrooved substrates versus 3D cell culture on flat substrates. As extension of our current BOC toolbox for which these indicative parameters would be used, the microtunnel devices show that culture of SH-SY5Y was feasible, though a limited number of neurites extended into microtunnels away from the cell bodies, regardless of using nanogrooved or flat substrates. This shows that the novel combination of microtunnel devices with nanogrooves can be implemented toward neuronal cell cultures, with future improvements to be performed to ensure neurites extend beyond the confines of the wells between the microtunnels. Overall, these results will aid toward creating more robust BOC platforms with improved predictive value. In turn, this can be used to better understand the brain and brain diseases.
3D-electrode integrated microsieve structure as a rapid and cost-effective single neuron detector
Retinoic Acid Accelerates the Specification of Enteric Neural Progenitors from In-Vitro-Derived Neural Crest
The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1 and 7. Defects in the development and function of the ENS cause a range of enteric neuropathies, including Hirschsprung disease. Little is known about the signals that specify early ENS progenitors, limiting progress in the generation of enteric neurons from human pluripotent stem cells (hPSCs) to provide tools for disease modeling and regenerative medicine for enteric neuropathies. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and, following in vivo transplantation, achieved long-term colonization of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.
Reproducible generation of human midbrain organoids for in vitro modeling of Parkinson’s disease
The study of human midbrain development and midbrain related diseases, like Parkinson’s disease (PD), is limited by deficiencies in the currently available and validated laboratory models. Three dimensional midbrain organoids represent an innovative strategy to recapitulate some aspects of the complexity and physiology of the human midbrain. Nevertheless, also these novel organoid models exhibit some inherent weaknesses, including the presence of a necrotic core and batch-to-batch variability. Here we describe an optimized approach for the standardized generation of midbrain organoids that addresses these limitations, while maintaining key features of midbrain development like dopaminergic neuron and astrocyte differentiation. Moreover, we have established a novel time-efficient, fit for purpose analysis pipeline and provided proof of concept for its usage by investigating toxin induced PD.
Nanogroove-enhanced hydrogel scaffolds for 3D neuronal cell culture: an easy access brain-on-chip model
In order to better understand the brain and brain diseases, in vitro human brain models need to include not only a chemically and physically relevant microenvironment, but also structural network complexity. This complexity reflects the hierarchical architecture in brain tissue. Here, a method has been developed that adds complexity to a 3D cell culture by means of nanogrooved substrates. SH-SY5Y cells were grown on these nanogrooved substrates and covered with Matrigel, a hydrogel. To quantitatively analyze network behavior in 2D neuronal cell cultures, we previously developed an automated image-based screening method. We first investigated if this method was applicable to 3D primary rat brain cortical (CTX) cell cultures. Since the method was successfully applied to these pilot data, a proof of principle in a reductionist human brain cell model was attempted, using the SH-SY5Y cell line. The results showed that these cells also create an aligned network in the 3D microenvironment by maintaining a certain degree of guidance by the nanogrooved topography in the z-direction. These results indicate that nanogrooves enhance the structural complexity of 3D neuronal cell cultures for both CTX and human SH-SY5Y cultures, providing a basis for further development of an easy access brain-on-chip model.
Read more https://www.mdpi.com/2072-666X/10/10/638
Exploiting nanogroove-induced cell culture anisotropy to advance in vitro brain models
A new generation of in vitro human brain models is vital to surpass the limitations of current cell culture platforms and animal cell lines in studying brain function and diseases. Brain-on-chip technology can generate well-defined and reproducible platforms to control the cellular microenvironment for in vivo-like, organized brain cell cultures. Previously, the authors investigated differentiation and network organization of the neuroblastoma SH-SY5Y cell line on nanogrooved substrates, showing that nanogroove guidance of neuronal outgrowths is dependent on nanogroove dimensions. Further, increased orientation of neurites was positively correlated to the differentiation of SH-SY5Y cells. However, as mimicking brain structure alone is insufficient, here, the function of the neuronal cell network as dependent on surface topography and material stiffness is investigated. A generalized replication protocol was developed to create similar nanogrooved patterns in cell culture substrates from different materials, specifically polydimethylsiloxane (PDMS) and Ostemer. Experiments using calcium imaging, where calcium fluxes across membranes are visualized as an indication of action potentials in neuronal cells, were performed with differentiated SH-SY5Y cells and human induced pluripotent stem cell-derived neuronal cells (hiPSCNs) on flat versus nanogrooved substrates to study the network function. Calcium live-imaging was performed and results for experiments with SH-SY5Y cells and hiPSCNs showed that nanogrooved PDMS substrates trended toward increased cellular activity and neuronal cell network connectivity. For future investigation of compatible substrate materials in combination with the effect of material stiffness on the cells, nanogrooved Ostemer substrates were demonstrated to faithfully replicate for use in neuronal cell cultures using nanogrooved substrates. First experiments into the neuronal cell function using stem cells described here aid toward elucidating the effect of nanotopographical and mechanical properties and their benefits toward advancing in vitro neuronal cell models both in form and function. Overall, the results indicate, in conjunction with the previous findings on neuronal outgrowth guidance, that anisotropy as introduced by nanogrooved substrates can have a controllable and potentially beneficial influence on neuronal cell cultures.
Retinoic acid accelerates the specification of enteric neural progenitors from in vitro-derived neural crest
The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1-7. Defects in the development and function of the ENS give rise to a range of disorders, termed enteric neuropathies and include conditions such as Hirschsprung’s disease. Little is known about the signalling that specifies early ENS progenitors. This has, thus far, limited progress in the generation of enteric neurons from human Pluripotent Stem Cells (hPSCs) that could provide a useful tool for disease modelling and regenerative medicine. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of both vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and following in vivo transplantation, achieving long-term colonisation of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS.