Name: preparation of tunable extracellular matrix microenvironments to evaluate schwann cell phenotype specification
Uploaded: 2020-06-02T14:30:00.000+00:00
Duration: 7 min 50 s
Description: Watch this Scientific Journal Video about Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification at JoVE.com

The authors gratefully acknowledge funding support from the University of Cincinnati. The authors also thank Ron Flenniken of the University of Cincinnati Advanced Materials Characterization laboratory for support.

No potential conflict of interest was reported by the authors.

SCs can promote nerve regeneration due to their phenotypic transformation and regenerative potential following nerve injury. However, how ECM cues regulate this regenerative capacity remains mostly unclear, potentially hindering not only the development of biomaterials that aim to promote nerve regeneration but also the understanding of the mechanisms involved in nerve regeneration. To begin to examine this interplay, cell culture substrates were created where ECM cues such as stiffness, protein coating, and adhesive topography can be controlled. The ability to micropattern adhesive topography is a key feature within the protocol, utilizing the common method of microcontact printing. However, this substrate is different from the glass in that the compression force being applied to the PDMS stamps must be at an appropriate level to achieve the desired shape of cell adhesive areas on cell culture substrates. Utilizing fluorescent bovine serum albumin (fBSA) as a model protein to visualize cell adhesive areas and adjust the compression forces on the PDMS stamps can ultimately mitigate this issue. Another key difference from glass is the removal of excess Pluronic F-127 that is used to render the remainder of the substrate non-adhesive. After this treatment, cell culture substrates are highly hydrophobic, making it challenging to maintain wet cell culture substrates throughout washes, which is important for the structural integrity of micropatterned protein37. Therefore, it is recommended to use multiple pipettes to aspirate and inject solutions nearly simultaneously to prevent substrates from completely dehydrating.
Although micropatterning can precisely control cell shape and does not require complicated synthetic procedures using cell blocking polymers such as OEGMA, the methods used to quantify protein expression of micropatterned cells are sometimes limited38. For example, samples that are available from micropatterning are generally limited to a few hundred cells per coverslip, which is inadequate to prepare cell lysates to be used for western blots or qPCR. Considering this, immunofluorescence staining alone was used to quantify protein expression for micropatterned cells, limiting the variety of proteins that can be quantified. To address this, we utilized line patterns to promote cell elongation for larger cell populations and successfully prepared cell lysates that were analyzed by western blot. Other methods such as applied electric fields or aligned electrospun fibers can also be applied to control the elongation of cell populations39,40. However, electric fields may not fit into all applications for NGCs since the conductivity of commonly used biomaterials such as collagen-based polymer and silk is very low28,41,42. In contrast, aligned electrospun nanofibers have been successfully implanted into NGCs to promote SC alignment, elongation and migration as well as neurite outgrowth43,44. It may also prove compelling to compare SC behavior on line-patterned substrates against those on substrates with aligned nanofibers, since lined patterns and aligned nanofibers are two of the most common guidance mechanisms incorporated into NGCs45.
The micropatterning protocol detailed utilizes PDMS coated coverslips as cell culture substrates, with a maximum surface Young&#8217;s modulus of 1119 kPa. Such stiffness mimics many tissue, yet it may not be possible to model osteogenesis of mesenchymal stem cells, which generally require the surface Young&#8217;s modulus to exceed 1 Gpa46. For such situations, glass is an alternative candidate, however the adsorption of Pluronic F-127 requires relatively high surface hydrophobicity which glass does not possess. To increase hydrophobicity, glass can be treated with dimethyl dichlorosilane in dichlorobenzene. Following this, UV-Ozone treatment can be used to increase hydrophilicity for microcontact printing47.
Ultimately, a cell culture platform was developed where ECM stimuli can be tuned individually with protein expression quantified. We have determined that SC regenerative capacity is promoted by certain mechanical and chemical ECM cues, which can subsequently provide inspiration in the future design of biomaterial applications such as NGCs and cell transplantation processes where ECM features may need to be optimized24. Nonetheless, tuning these ECM cues can be a challenging undertaking, particularly in vivo. Moving forward, this platform can be utilized to parse key mechanisms involved in the phenotypic transition of SCs as regulated by the ECM. By achieving this, manipulation of intracellular cues may be possible to promote SC regenerative capacity without the need for dedicated platforms in vitro48,49. This has the potential for groundbreaking work in the development of technologies for nerve repair.

Peripheral nervous system (PNS) injuries remain a major clinical challenge in healthcare by compromising the quality of life for patients and creating a significant impact through a multitude of socioeconomic factors1,2. Schwann cells (SC), as the major glial cells in the PNS, provide necessary molecular and physical cues to induce PNS regeneration and aid in functional recoveries in short gap injuries. This is due to the remarkable ability of SCs to dedifferentiate into a &#8220;repair&#8221; cell phenotype from a myelinating or Remak phenotype3. The repair SC is a distinctive cell phenotype in several ways. Following injury, SCs increase their proliferation rate by re-entering the cell cycle and begin expression of several transcriptional factors to facilitate reinnervation. These factors, such as c-Jun and p75 NTR, are upregulated while myelinating SC markers, such as myelin basic protein (MBP), are downregulated4,5. In addition, SCs change morphology to become elongated and aligned with each other to form B&#252;ngner bands across the injury site6. This provides a physical guidance mechanism for the axons to extend to the correct distal target7. However, despite the ability that SCs possess to promote nerve regeneration in short gap injuries, the outcome of functional recovery remains poor in severe injuries. This is due in part to a loss of extracellular matrix (ECM) guidance cues, as well as the inability of SCs to maintain the regenerative phenotype over long periods of time8.
The nerve regeneration and recovery process are intimately tied to the state of the basal lamina following injury. The basal lamina is a layer of ECM around the nerve that facilitates guidance and provides ECM-bound cues for axons and SCs in cases where it remains intact following injury9. The state of the ECM and its ability to deliver matrix bound cues to cells is vitally important and has been previously explored in a variety of different contexts10,11,12,13,14. For example, it has been shown that the stiffness of the ECM can guide cell functions such as proliferation and differentiation11,15,16. Composition of the ECM can also lead to a distinct cellular response and regulate cell behaviors such as migration and differentiation through intracellular signaling pathways17,18. Furthermore, cell morphology, including spreading area and cellular elongation, play a major role in regulating the function and can be governed by ECM-bound cues19,20. Many previous studies have focused on stem cells differentiating into defined lineages, yet SCs possess a similar ability to alter phenotype from a homeostatic, adult SC within a healthy nerve, to a repair SC capable of secreting proteins and growth factors while remodeling the ECM following nerve injury5,21. Therefore, it is especially crucial to identify mechanisms underlying the relationship between the innate SC regenerative capacity and ECM bound cues for the insight to ultimately harness this capacity for nerve regeneration.
To address this, we have developed a detailed methodology to produce a cell culture substrate where mechanical stiffness and ligand type can be easily tuned in physiologically relevant ranges. Polydimethyl siloxane (PDMS) was chosen as a substrate due to its highly tunable mechanics as compared to polyacrylamide gel, where the maximum Young&#8217;s modulus is around 12 kPa contrasted to PDMS at around 1000 kPa22,23,24. This is beneficial to the work at hand, as recent studies have shown the Young&#8217;s modulus of a rabbit sciatic nerve can exceed 50 kPa during development, thereby suggesting that the range of stiffness of nerves within the PNS is wider than previously examined. Different proteins are capable of adsorption onto PDMS substrates to analyze the combinatorial regulation of mechanics and ligands on SC behavior. This allows for the investigation of multiple microenvironmental cues present in the PNS regeneration process and comparison of a high degree of tunability to the work focusing solely on stiffness of the substrate25. Further, these engineered cell culture substrates are compatible with a multitude of quantitative analysis methods such as immunohistochemistry, western blot, and quantitative polymerase chain reaction (q-PCR).
This engineered cell culture platform is highly suitable for analyzing mechanistic pathways due to the high level of individual tunability of each ECM-bound signal. In addition, popular methods for cell micropatterning, including microcontact printing, can be achieved on the substrates to allow for controlled cellular adhesion to analyze cell shape in relation to other ECM bound cues24. This is critical because line patterned substrates, which promote elongation in cell populations, provide a tool to mimic and study elongated and regenerative SCs within B&#252;ngner bands during nerve regeneration. Further, cellular morphology is a potent regulator of multiple cell functions and can potentially introduce confounding experimental results if not controlled26,27. Significant attention is now being provided to the mechanisms governing the SC regenerative phenotype as regulated by ECM cues28,29,30. This is essential to provide insight into the design of biomaterials that can be applied as nerve guidance conduits for aid in PNS nerve regeneration. These detailed protocols can ultimately be applied as a potential tool to decipher the mechanisms of SC and other cell type function as regulated by ECM bound cues.

<table><tbody><tr><td>Albumin from Bovine Serum (BSA), Texas Red conjugate</td><td>Thermo Fisher Scientific</td><td>A23017</td><td>BSA staining to show micropatterns</td></tr><tr><td>Anti-mouse IgG, HRP-linked Antibody</td><td>Cell Signaling Technology</td><td>7076S</td><td>Antibody used for western blot analysis</td></tr><tr><td>Anti-rabbit IgG, HRP-linked Antibody</td><td>Cell Signaling Technology</td><td>7074S</td><td>Antibody used for western blot analysis</td></tr><tr><td>BrdU</td><td>Thermo Fisher Scientific</td><td>B23151</td><td>Reagent used to measure cell proliferation</td></tr><tr><td>BrdU primary antibody conjugated with Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>B35130</td><td>Used to visualize BrdU in cell proliferation assays</td></tr><tr><td>Collagen I</td><td>Thermo Fisher Scientific</td><td>A10483-01</td><td>Protein used to coat coverslips</td></tr><tr><td>Compression force test machine</td><td>TestResources</td><td></td><td>Instrument to quantify mechanical properties of polymers</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium</td><td>Thermo Fisher Scientific</td><td>11965092</td><td>Cell culture medium</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermo Fisher Scientific</td><td>16000044</td><td>Cell culture medium supplemental</td></tr><tr><td>Fibronectin</td><td>Thermo Fisher Scientific</td><td>33010-018</td><td>Protein used to coat coverslips</td></tr><tr><td>Fluorescence microscope</td><td>Nikon</td><td>Eclipse Ti2</td><td>Fluorescence microscope</td></tr><tr><td>Halt Protease and Phosphatase Inhibitor Cocktail (100X)</td><td>Thermo Fisher Scientific</td><td>78440</td><td>Protease and Phosphatase Inhibitor</td></tr><tr><td>Laminin</td><td>Thermo Fisher Scientific</td><td>23017015</td><td>Protein used to coat coverslips</td></tr><tr><td>Mounting medium with DAPI</td><td>Thermo Fisher Scientific</td><td>P36971</td><td>Coverslip mountant and nuclei staining</td></tr><tr><td>Mouse c-Jun primary antibody</td><td>Thermo Fisher Scientific</td><td>711202</td><td>Primary antibody to visualize c-Jun protein</td></tr><tr><td>Mouse &amp;beta;-Actin primary antibody</td><td>Cell Signaling Technology</td><td>3700S</td><td>Loading control for western blot experiments</td></tr><tr><td>Penicillin-Streptomycin</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Cell culture medium supplemental</td></tr><tr><td>Photoresist SU 2010</td><td>KAYAKU</td><td>SU8-2010</td><td>Photoresist</td></tr><tr><td>Pluronic F-127</td><td>Sigma Aldrich</td><td>P-2443</td><td>Block non-specific protein binding</td></tr><tr><td>Rabbit c-Jun primary antibody</td><td>Cell Signaling Technology</td><td>9165S</td><td>Primary antibody for visualization of c-Jun protein</td></tr><tr><td>Rabbit myelin basic protein primary antibody</td><td>Abcam</td><td>ab40390</td><td>Primary antibody for visualization of MBP</td></tr><tr><td>Rabbit p75NTR primary antibody</td><td>Cell Signaling Technology</td><td>8238S</td><td>Primary antibody for visualization of p75NTR</td></tr><tr><td>Rhodamine phalloidin</td><td>Thermo Fisher Scientific</td><td>R415</td><td>Visualization of cell cytoskeleton</td></tr><tr><td>RIPA buffer</td><td>Abcam</td><td>ab156034</td><td>Cell lysis buffer</td></tr><tr><td>RT4-D6P2T Schwann cell line</td><td>ATCC</td><td>CRL-2768</td><td>Cell line used in experiments</td></tr><tr><td>SYLGARD 184 PDMS base and curing agent</td><td>Sigma Aldrich</td><td>761036</td><td>Tunable polymer used to coat coverslips</td></tr><tr><td>Trypsin</td><td>Thermo Fisher Scientific</td><td>15090-046</td><td>Cell dissociation reagent</td></tr><tr><td>UV-Ozone cleaner</td><td>Novascan</td><td></td><td>Increase hydrophicility of PDMS</td></tr><tr><td>Versene (1x)</td><td>Thermo Fisher Scientific</td><td>15040066</td><td>Cell dissociation reagent</td></tr></tbody></table>

preparation of tunable extracellular matrix microenvironments to evaluate schwann cell phenotype specification

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the major glial cells of the PNS, play a vital role in promoting PNS regeneration by dedifferentiating into a regenerative cell phenotype following injury. However, the dedifferentiated state of SCs is challenging to maintain through the time-period needed for regeneration and is impacted by changes in the surrounding extracellular matrix (ECM). Therefore, determining the complex interplay between SCs and differing ECM to provide cues of regenerative potential of SCs is essential. To address this, a strategy was created where different ECM proteins were adsorbed onto a tunable polydimethylsiloxane (PDMS) substrate which provided a platform where stiffness and protein composition can be modulated. SCs were seeded onto the tunable substrates and critical cellular functions representing the dynamics of SC phenotype were measured. To illustrate the interplay between SC protein expression and cellular morphology, differing seeding densities of SCs in addition to individual microcontact printed cellular patterns were utilized and characterized by immunofluorescence staining and western blot. Results showed that cells with a smaller spreading area and higher extent of cellular elongation promoted higher levels of SC regenerative phenotypic markers. This methodology not only begins to unravel the significant relationship between the ECM and cellular function of SCs, but also provides guidelines for the future optimization of biomaterials in peripheral nerve repair.

To analyze and quantify the interplay between substrate stiffness and protein composition on SC phenotype, a tunable PDMS cell culture substrate was developed (Figure 1A). Compression testing of the polymer at differing base: curing agent ratios was utilized to quantify the Young&#8217;s modulus (E) of the substrate (Figure 1B). The resulting range of modulus values represents physiologically relevant substrate conditions. Following preparation of substrates, SCs were cultured and cellular properties analyzed on the tunable microenvironment. Proliferation rates of SCs on substrates of differing protein composition were first analyzed. Laminin coated substrates resulted in a higher proliferation rate when compared to collagen I and fibronectin adsorption all at 10 &#181;g/mL (Figure 2A,B). SCs on substrates with laminin coating and differing moduli showed that relatively softer substrates (E=3.85kPa) decrease cell proliferation rates across all conditions (Figure 2C,D). However, differences between stiff substrates (E=1119kPa) and relatively soft substrates (E=8.67kPa) were insignificant (Figure 2D).
SCs were also analyzed for protein expression through immunohistochemistry and western blot. Levels of transcriptional factor c-Jun were analyzed by immunofluorescent microscopy (Figure 3A) and represented by the mean pixel fluorescent intensity (Figure 3B-D). c-Jun expression was shown to be upregulated as substrates became softer (E=1119 kPa to E=8.67 kPa), however, on the softest substrates (E=3.85 kPa), c-Jun expression was significantly downregulated. On stiff substrates (E=1119 kPa), collagen I coated substrates resulted in the highest c-Jun expression, yet as substrates became softer (E=8.67 kPa and 3.85 kPa), laminin showed the highest levels of c-Jun (Figure 3E). Western blot was also used to analyze both c-Jun and myelin basic protein (MBP) with c-Jun levels upregulated and MBP downregulated on softer substrates (Figure 3F). Further, SCs seeded on laminin coated substrates resulted in the highest c-Jun expression when compared to collagen I and fibronectin.
Cells of differing seeding densities were then cultured and stained with rhodamine-phalloidin to explore the role of cell spreading and area in c-Jun expression (Figure 4A,B). To control nuclear elongation of cells, a common micropatterning technique (microcontact printing36) was utilized to create cell adhesive lines on the cell culture substrates. The nuclear aspect ratio of cells seeded on a line patterned substrate was shown to be significantly higher than cells seeded on unpatterned substrates (Figure 4C,D). It was found that expression of both c-Jun and another marker significant in SC regenerative phenotypes, p75 neurotrophin receptor (p75 NTR), were upregulated in dense cells with a smaller spreading area (Figure 4E). Line-patterned cells also resulted in a higher expression of both c-Jun and p75 NTR when compared to nonpatterned cells (Figure 4F). Therefore, microcontact printed cell adhesive geometries were created to precisely control cell spreading area and elongation while eliminating cell-cell interactions (Figure 5A). Dimensions of total cell adhesive areas were 900 &#181;m2, 1,600 &#181;m2, and 2,500 &#181;m2 with an aspect ratio of either 1 or 4 (cell length: cell width). Fluorescent bovine serum albumin (fBSA, Texas red) staining was used to reveal the status of micropatterns on cell culture substrate after microcontact printing (Figure 5B). The nuclear aspect ratio of SCs for each cell area was measured and it was shown that by increasing the nuclear aspect ratio, the cellular elongation increases (Figure 5C). In addition, as SC aspect ratio increased, c-Jun was upregulated (Figure 5D). Interestingly, however, it was found that as cell spreading area increases c-Jun expression is downregulated (Figure 5E). Immunofluorescence staining for both nuclei and actin confirmed cell spreading area and elongation were highly controlled through this micropatterning method (Figure 5F).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61496/61496fig01.jpg" /> 
Figure 1: Cell culture substrates with tunable stiffness and protein composition.&#160;(A) Schematic showing the development of PDMS cell culture substrates. (B) The initial PDMS mixing ratio of base: curing agent determines Young&#8217;s modulus. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61496/61496fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61496/61496fig02.jpg" /> 
Figure 2: SC proliferation rates regulated by substrate stiffness and protein composition.&#160;(A) Representative images showing BrdU staining when cultured on substrates of the same modulus. (B) Histogram showing the percentage of BrdU positive cells for each protein coating. (C) Representative images showing BrdU incorporation in SCs seeded on substrates of the same protein coating. (D) Histogram showing the percentage of BrdU positive cells for each Young&#8217;s modulus value. Scale bars = 50 &#956;m. Data are presented as mean &#177; SEM. *p &#60; .05, **p &#60; .005, ***p &#60; .0005. Portions of the figure have been modified from ref.24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61496/61496fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61496/61496fig03.jpg" /> 
Figure 3: Substrate stiffness and protein regulated SC protein expression.&#160;(A) Representative images show c-Jun immunofluorescence staining for SCs seeded on substrates of different stiffness and protein composition. Mean pixel fluorescent intensity of c-Jun was measured for SCs seeded on (B) collagen I (C) fibronectin and (D) laminin coated substrates of different stiffness. (E) c-Jun fluorescence level of SCs grouped by Young&#8217;s modulus of substrate. (F) Western blot showing c-Jun and myelin basic protein (MBP) of SCs seeded on substrates. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. Scale bar = 50 &#956;m. Data are presented as mean &#177; SEM. *p &#60; .05, **p &#60; .005, ***p &#60; .0005. Portions of the figure have been modified from ref.24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61496/61496fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61496/61496fig04.jpg" /> 
Figure 4: Cellular spreading area influences protein expression of SCs.&#160;(A) Cell spreading area of different seeding densities was visualized through rhodamine-phalloidin (red) and nuclei staining (blue). (B) Histogram showing the average spreading area of SCs in each condition. (C) The nuclei of SCs seeded on unpatterned or line-patterned substrates were stained with DAPI (blue) to show morphology. (D) Histogram showing quantification of nuclear aspect ratio on patterned and unpatterned substrates. (E) Western blot showing expression of c-Jun and p75NTR of cells with different spreading area. (F) Western blot showing the protein expression of SCs on unpatterned and line-patterned substrates. Scale bar = 50 &#956;m. Data are presented as mean &#177; SEM. *p &#60; .05, **p &#60; .005, ***p &#60; .0005. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61496/61496fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61496/61496fig05.jpg" /> 
Figure 5: SC morphology and elongation impact c-Jun expression of SCs.&#160;(A) Schematic showing cell micropatterning for shapes of different aspect ratio. (B) fBSA staining (red) showing the shape of micropatterns following microcontact printing. Scale bar = 10 &#956;m. (C) Histogram showing nuclear aspect ratio of micropatterned SCs. (D, E) Histogram showing the mean pixel fluorescent intensity of c-Jun for each of the geometrical conditions. (F) Rhodamine-phalloidin (red), nuclei (blue) and c-Jun (green) were stained on the differing micropatterns. Scale bar = 10 &#956;m. Data are presented as mean &#177; SEM. *p &#60; .05, **p &#60; .005, ***p &#60; .0005. Portions of the figure have been modified from ref.24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61496/61496fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification at JoVE.com

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

This methodology aims to illustrate the mechanisms by which extracellular matrix cues such as substrate stiffness, protein composition and cell morphology regulate Schwann cell (SC) phenotype.

Traumatic peripheral nervous system (PNS) injuries currently lack suitable treatments to regain full functional recovery. Schwann cells (SCs), as the ...

preparation-tunable-extracellular-matrix-microenvironments-to

Nanyang Technological University

Research

JoVE Journal

Bioengineering

5.2K Views.  University of Cincinnati. Dit protocol biedt een celcultuurplatform waar de eigenschappen van extracellulaire matrix zoals stijfheid, eiwitsamenstelling en celmorfologie nauwkeurig kunnen worden gecontroleerd op een kosteneffectieve en facile manier. De methode is kosteneffectief en vereist geen speciale training of faciliteiten. Het is ook compatibel met meerdere cellulaire kwantificering methoden zoals immunofluorescente vlekken en westerse vlek.Deze techniek biedt een mechanistisch begrip van Schwann celbiol...

Video: Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

Developmental Biology

Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the mature Schwann cell fate. We subjected rat and human MSCs to transient hypoxic conditions (1% oxygen for 16 h) followed by expansion as neurospheres upon low-attachment substratum with epidermal growth factor (EGF)/basic fibroblast growth factor (bFGF) supplementation. Neurospheres were seeded onto poly-D-lysine/laminin-coated tissue culture plastic and cultured in a gliogenic cocktail containing &#946;-Heregulin, bFGF, and platelet-derived growth factor (PDGF) to generate Schwann cell-like cells (SCLCs). SCLCs were directed to fate commitment via coculture for 2 weeks with purified dorsal root ganglia (DRG) neurons obtained from E14-15 pregnant Sprague Dawley rats. Mature Schwann cells demonstrate persistence in S100&#946;/p75 expression and can form myelin segments. Cells generated in this manner have potential applications in autologous cell transplantation following spinal cord injury, as well as in disease modeling.

Marrow stromal cells (MSCs) with neural potential exist within the bone marrow. Our protocol enriches this population of cells via hypoxic preconditioning and thereafter directs them to become mature Schwann cells.

This manuscript describes a means to enrich for neural progenitors from the marrow stromal cell (MSC) population and thereafter to direct them to the ...

Analyzing Murine Schwann Cell Development Along Growing Axons

The development of peripheral nerves is an intriguing process. Neurons send out axons to innervate specific targets, which in humans are often more than 100 cm away from the soma of the neuron. Neuronal survival during development depends on target-derived growth factors but also on the support of Schwann cells (SCs). To this end SC ensheath axons from the region of the neuronal soma (or the transition from central to peripheral nervous system) to the synapse or neuromuscular junction. Schwann cells are derivatives of the neural crest and migrate as precursors along emerging axons until the entire axon is covered with SCs. This shows the importance of SC migration for the development of the peripheral nervous system and underlines the necessity to investigate this process. In order to analyze SC development, a setup is needed which next to the SCs also includes their physiological substrate for migration, the axon. Due to intrauterine development in vivo time-lapse imaging, however, is not feasible in placental vertebrates like mouse (mus musculus). To circumvent this, we adapted the superior cervical ganglion (SCG) explant technique. Upon treatment with nerve growth factor (NGF) SCG explants extend axons, followed by SC precursors migrating along the axons from the ganglion to the periphery. The beauty of this system is that the SC are derived from a pool of endogenous SC and that they migrate along their own physiological axons which are growing at the same time. This system is especially intriguing, because the SC development along axons can be analyzed by time-lapse imaging, opening further possibilities to gain insights into SC migration.

Here we describe a Schwann cell (SC) migration assay in which SCs are able to develop along extending axons.

The  development of peripheral nerves is an intriguing process. Neurons send out  axons to innervate specific targets, which in humans are often more than ...

Neuroscience

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5.
However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13.
In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour.
These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries.
The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip.
Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19.
This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using In Vitro Assays

This article intends to describe in stepwise fashion the commonly used in vitro assays used in studying Schwann cell-asrtocyte interactions.

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal ...

Biology

Anatomically Inspired Three-dimensional Micro-tissue Engineered Neural Networks for Nervous System Reconstruction, Modulation, and Modeling

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory environment and the limited capacity for neurogenesis. We are developing&#160;a strategy to simultaneously address&#160;neuronal and axonal pathway loss within the damaged CNS. This manuscript presents the fabrication protocol for micro-tissue engineered neural networks (micro-TENNs), implantable constructs consisting of neurons and aligned axonal tracts spanning the extracellular matrix (ECM) lumen of a preformed hydrogel cylinder hundreds of microns in diameter that may extend centimeters in length. Neuronal aggregates are delimited to the extremes of the three-dimensional encasement and are spanned by axonal projections. Micro-TENNs are uniquely poised as a strategy for CNS reconstruction, emulating aspects of brain connectome cytoarchitecture and potentially providing means for network replacement. The neuronal aggregates may synapse with host tissue to form new functional relays to restore and/or modulate missing or damaged circuitry. These constructs may also act as pro-regenerative&#160;&#34;living scaffolds&#34; capable of exploiting developmental mechanisms for cell migration and axonal pathfinding, providing synergistic structural and soluble cues based on the state of regeneration. Micro-TENNs are fabricated by pouring liquid hydrogel into a cylindrical mold containing a longitudinally centered needle. Once the hydrogel has gelled, the needle is removed, leaving a hollow micro-column. An ECM solution is added to the lumen to provide an environment suitable for neuronal adhesion and axonal outgrowth. Dissociated neurons are mechanically aggregated for precise seeding within one or both ends of the micro-column. This methodology reliably produces self-contained miniature constructs with long-projecting axonal tracts that may recapitulate features of brain neuroanatomy. Synaptic immunolabeling and genetically encoded calcium indicators suggest that micro-TENNs possess extensive synaptic distribution and intrinsic electrical activity. Consequently, micro-TENNs represent a promising strategy for targeted neurosurgical reconstruction of brain pathways and may also be applied as biofidelic models to study neurobiological phenomena in vitro.

This manuscript details the fabrication of micro-tissue engineered neural networks: three-dimensional micron-sized constructs comprised of long aligned axonal tracts spanning aggregated neuronal population(s) encased in a tubular hydrogel. These living scaffolds can serve as functional relays to reconstruct or modulate neural circuitry or as biofidelic test-beds mimicking gray-white matter neuroanatomy.

Functional recovery rarely occurs&#160;following injury or disease-induced degeneration within the central nervous system (CNS)&#160;due to the inhibitory ...

Fabrication and Use of MicroEnvironment microArrays (MEArrays)

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, distinct microenvironments can impose differentiation, migration, and proliferation phenotypes, and on the tissue level the microenvironment processes as complex as morphogenesis and tumorigenesis1. Not only do the cell and molecular contents of microenvironments impact the cells within, but so do the elasticity2 and geometry3 of the tissue. Defined as the sum total of cell-cell, -ECM, and -soluble factor interactions, in addition to physical characteristics, the microenvironment is complex. The phenotypes of cells within a tissue are partially due to their genomic content and partially due to the combinatorial interactions with the microenviroment. A major challenge is to link specific combinations of microenvironmental components with distinctive behaviours.
Here, we present the microenvironment microarray (MEArray) platform for cell-based functional screening of interactions with combinatorial microenvironments4. The method allows for simultaneous control of the molecular composition and the elastic modulus, and combines the use of widely available microarray and micropatterning technologies. MEArray screens require as few as 10,000 cells per array, which facilitates functional studies of rare cell types such as adult progenitor cells. A limitation of the technology is that entire tissue microenvironments cannot be completely recapitulated on MEArrays. However, comparison of responses in the same cell type to numerous related microenvironments, for instance pairwise combinations of ECM proteins that characterize a given tissue, will provide insights into how microenvironmental components elicit tissue-specific functional phenotypes.
MEArrays can be printed using a wide variety of recombinant growth factors, cytokines, and purified ECM proteins, and combinations thereof. The platform is limited only by the availability of specific reagents. MEArrays are amenable to time-lapsed analysis, but most often are used for end point analyses of cellular functions that are measureable with fluorescent probes. For instance, DNA synthesis, apoptosis, acquisition of differentiated states, or production of specific gene products are commonly measured. Briefly, the basic flow of an MEArray experiment is to prepare slides coated with printing substrata and to prepare the master plate of proteins that are to be printed. Then the arrays are printed with a microarray robot, cells are allowed to attach, grow in culture, and then are chemically fixed upon reaching the experimental endpoint. Fluorescent or colorimetric assays, imaged with traditional microscopes or microarray scanners, are used to reveal relevant molecular and cellular phenotypes (Figure 1).

Fabrication and Use of MicroEnvironment microArrays MEArrays

A combinatorial functional screening method for gaining insights into the impacts of the molecular composition of microenvironments on cellular functions is described. The method takes advantage of existing microarray-based technologies to generate arrays of defined combinatorial microenvironments that support cell adhesion and functional analysis.

The interactions between cells and their surrounding microenvironment have functional consequences for cellular behaviour. On the single cell level, ...

Generation of 3-D Collagen-based Hydrogels to Analyze Axonal Growth and Behavior During Nervous System Development

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is centered on culturing small pieces of embryonic or early postnatal rodent brains inside a 3-D hydrogel formed by the rat tail tendon-derived type I collagen with specific porosity. Tissue pieces are cultured inside the hydrogel and confronted to specific brain fragments or genetically-modified cell aggregates to produce and secrete molecules suitable for creating a gradient inside the porous matrix. The steps of this protocol are simple and reproducible but include critical steps to be considered carefully during its development. Moreover, the behavior of growing axons can be monitored and analyzed directly using a phase-contrast microscope or mono/multiphoton fluorescence microscope after fixation by immunocytochemical methods.

Here, we provide a method for analyzing the behavior of growing axons in 3D matrices, mimicking their natural development.

This protocol uses natural type I collagen to generate three-dimensional (3-D) hydrogel for monitoring and analyzing the axonal growth. The protocol is ...

Generating Schwann Cell-Like Cells from Bone Marrow Stromal Cells

Source: Tsui, Y., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/55794">Hypoxic Preconditioning of Marrow-derived Progenitor Cells As a Source for the Generation of Mature Schwann Cells.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method for obtaining Schwann cell-like cells from multipotent bone marrow stromal cells (BMSCs). Initially, the BMSCs undergo hypoxia treatment, followed by directed differentiation into neuronal progenitor cells. Subsequently, specific media and culture conditions are employed to induce the transformation of these progenitor cells into Schwann cell-like cells.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparation of Rat MSC Cultures

	...

JoVE Encyclopedia of Experiments

Neuronal Culture Techniques

Advanced Neuronal Models

Co-Culture of Schwann Cells with DRG Neurons on a Pre-Stretched Membrane

Source: Liu, C., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/54085">An Approach to Enhance Alignment and Myelination of Dorsal Root Ganglion Neurons.</a> J. Vis. Exp. (2016).
The video demonstrates the co-culturing of dorsal root ganglion (DRG) neurons with Schwann cells. DRG neurons are first grown on a pre-stretched membrane for aligned axon growth. Schwann cells are then introduced, which attach to the axons, insulating them and promoting neural cell survival and function.

1. Culture of dorsal root ganglion (DRG) on Pre-stretched Surface

	Preparation of Pre-stretched Anisotropic Surface
	
		Mix a 10:1 solution of base and ...

Co-culture and Interaction Studies

Preparation of a Micropatterned Substrate to Study Schwann Cell Phenotypes

Source: Xu, Z., Orkwis, et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/61496">Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification.</a> J. Vis. Exp. (2020).
The video describes the preparation of a micropatterned substrate to study cell phenotype specification. A protein-coated micropatterned PDMS stamp is used to transfer the micropattern on a PDMS-coated coverslip. The coverslip is then treated with a surfactant to hinder cell adhesion at the unwanted surfaces. Cells are then added to settle in the pattern and incubated for further analysis.

1. Tunable cell culture substrate preparation and characterization

	Substrate preparation&#8203;
	
		Mix the Polydimethylsiloxane (PDMS) base elastomer ...

Preparation of Tunable Extracellular Matrix Microenvironments to Evaluate Schwann Cell Phenotype Specification

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