A neural extracellular matrix-based method for in vitrohippocampal neuron culture and dopaminergic differentiation of neural stem cells
© García-Parra et al.; licensee BioMed Central Ltd. 2013
Received: 8 January 2013
Accepted: 13 April 2013
Published: 18 April 2013
The ability to recreate an optimal cellular microenvironment is critical to understand neuronal behavior and functionality in vitro. An organized neural extracellular matrix (nECM) promotes neural cell adhesion, proliferation and differentiation. Here, we expanded previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following goals: (i) to recreate complex neuronal networks of embryonic rat hippocampal cells, and (ii) to achieve improved levels of dopaminergic differentiation of subventricular zone (SVZ) neural progenitor cells.
Hippocampal cells from E18 rat embryos were seeded on PLL- and nECM-coated substrates. Neurosphere cultures were prepared from the SVZ of P4-P7 rat pups, and differentiation of neurospheres assayed on PLL- and nECM-coated substrates.
When seeded on nECM-coated substrates, both hippocampal cells and SVZ progenitor cells showed neural expression patterns that were similar to their poly-L-lysine-seeded counterparts. However, nECM-based cultures of both hippocampal neurons and SVZ progenitor cells could be maintained for longer times as compared to poly-L-lysine-based cultures. As a result, nECM-based cultures gave rise to a more branched neurite arborization of hippocampal neurons. Interestingly, the prolonged differentiation time of SVZ progenitor cells in nECM allowed us to obtain a purer population of dopaminergic neurons.
We conclude that nECM-based coating is an efficient substrate to culture neural cells at different stages of differentiation. In addition, neural ECM-coated substrates increased neuronal survival and neuronal differentiation efficiency as compared to cationic polymers such as poly-L-lysine.
KeywordsNeural extracellular matrix Subventricular zone Neuronal culture Neural progenitor cells Dopaminergic differentiation
The extracellular matrix (ECM) is responsible for the promotion of cell adhesion, proliferation and differentiation, and hence maintenance of tissue homeostasis throughout the whole organism. In the nervous system, neural ECM (nECM) components provide the necessary cues for the growth of neurons and neurites both in vitro and in vivo. Even during embryonic development, nECM plays a major role in the formation and expansion of the neural crest [2, 3]. Although mature nECM is amorphous, it also contains dense areas around neurons known as perineural networks (PNNs), which in turn leave spaces at the sites where synaptic contacts are established [4, 5]. PNNs are mainly composed of proteoglycans, collagen, laminin, fibronectin and neuropeptides. The latter seem to be involved in the regulation of neuronal plasticity  as well as in neuroprotection .
Topographical and biochemical characteristics of the substrate might thus be critical for axonal outgrowth and regeneration of neural circuits in vivo. For instance, cell therapy trials of Parkinson’s disease indicate that the mere existence of a damaged microenvironment promotes the generation of new dopaminergic neurons, although perhaps in numbers insufficient for clinical improvement .
In the adult central nervous system (CNS), neurogenesis seems to be restricted to two specific areas: (i) the subventricular zone (SVZ) [9, 10] and (ii) the subgranular layer of the dentate gyrus of the hippocampus [11, 12]. Neural stem cells (NSCs) with multipotent potential can be derived from rodent SVZ [10, 11, 13]. In vitro, the differentiation of NSCs into neuronal or glial cells is induced in adherent culture conditions (with serum) after EGF and FGF2 withdrawal and by specific neuronal- and/or glial-inducing factors. As the differentiation process progresses, NSCs restrict their ability to self-renew and differentiate to other lineages.
In a previous article we described the development, characterization and functional validation of a nECM-based polymeric support used as a biocompatible and efficient in vitro microenvironment for neuronal differentiation of both PC12 cells and adult skin-derived precursor cells . Since nECM proteoglycans are involved in axonal regeneration and in post-traumatic neuronal plasticity in the CNS [15, 16], the rationale for the present methodological article was to use the nECM component of this polymeric support to study neuronal development and, in particular, dopaminergic differentiation. The presence of collagen, laminin, entactin and certain proteoglycans in the basal composition of nECM provides the signals that cells need to be anchored to the substrate and start differentiation. Moreover, hyaluronic acid enhances the hydration of the matrix, which in turn will facilitate cell movement and neurite extension along the neuronal differentiation progress as well as provide fixation sites for growth factors  and other glycosaminoglycan-binding molecules . Finally, netrins support neuronal differentiation acting as cellular and neurite-chemoattractant factors, as it occurs in the naturally developing neural tissue .
As of today, several types of polymeric supports have been described to sustain neural cell adhesion and migration [20, 21]. Recent works demonstrated that polymeric substrates sustain differentiation of stem/progenitor cells and help generate functional synapses because the spatial, mechanical and biochemical cues that neurons receive from the environment strongly determine their in vitro behaviour [22–25]. Nevertheless, these studies also suggest the importance of soluble factors released from the ECM. Therefore, culturing stem/progenitor cells on three-dimensional ECMs could be a successful approach to maintain multipotent progenitor cell cultures for longer in vitro, as well as to achieve higher percentages of neuronal differentiation. In the present work, we studied biocompatibility and neuronal differentiation of primary neural cells at different stages of development on nECM-coated substrates. We were particularly interested in improving primary neuronal cultures of embryonic rat hippocampal cells and to obtain higher levels of dopaminergic neurons from neonatal rat SVZ multipotent progenitor cells, as compared to more “classical” cell culture conditions such as poly-L-lysine (PLL) coating.
Results and discussion
Culture of rat hippocampal cells on neural extracellular matrix (nECM)-coated surfaces
With regard to neuronal sprouting, after 24 and 72 h of culture on nECM, significant increases in the percentage of neurons sprouting neurites compared with their PLL counterparts were detected (Figure 2D). Moreover, these neurons extended a significantly higher total number of neurites (p < 0.001; Figure 2E) and a significantly higher (p < 0.001) number of neurites per neuron (4.8 ± 0.8 vs 2.8 ± 0.4 neurites per neuron at day 3, respectively; Figure 2F) than those on PLL-coated coverslips. Finally, nECM-based cultures were maintained for up to 30 days with cell media changed every 7 days (as compared to 2-day media changes in the case of PLL; data not shown). These data might support a role of netrins in cell survival as previously suggested by Tang and colleagues . Moreover, since netrins are known to induce neuronal sprouting during embryonic development, this effect might also be observed in the adult and partially help to explain the differences in neurite number in the presence (nECM) or absence (PLL) of added netrins. Additionally, cationic polymers such as PLL may be easily digested by cells, whereas a natural and more complex polymeric support such as nECM should be more efficient in sustaining cell adhesion and, consequently, might improve cell survival overall.
Once tested in isolation, the ability of CS-incorporated nECM to protect neuronal culture from cytotoxic damage (or otherwise) was analyzed. To this end, known cytotoxic compounds such as glutamate, beta-amyloid 25:35 peptide (Aβ) and oligomycin-rotenone (O/R) were added to cultures grown on nECM in the absence or presence of previously tested CS concentrations (Figure 3C-E). Higher concentration of CS significantly (P < 0.01) increased viability of neurons that were treated with 100 μM glutamate for 24 h (Figure 3C). The same neuroprotective tendency of CS was observed for Aβ and O/R treatments, but no statistical significance was achieved even at higher CS dosing (Figure 3D-E). Thus, in the presence of a cytotoxic agent as glutamate, a molecule liberated by injured cells in the central nervous system, CS (and probably other proteoglycans present in nECM) seemed to play a neuroprotective role, reversing the reduction of cellular viability seen when cells were seeded onto control nECM. Its apparent neuroprotective effect may be explained due to the known capacity of chondroitin 4-sulfate to sequester toxic compounds added to the medium. Alternatively, CS might increase the expression of antioxidant enzymes  or modulate the immuno-inflammatory response . These observations should be studied in more detail because they could have effects on promoting repair following spinal cord injury when the ECM changes its composition and expression of CS proteoglycans is augmented . Following chondroitinase ABC treatment, many groups have reported some improvement in spinal cord injury models and in vitro neuronal cultures recreating glial scar ECM [35–37].
Dopaminergic differentiation of subventricular zone (SVZ)-derived multipotent progenitor cells on nECM-coated surfaces
The effect of nECM on neurosphere differentiation was evident as early as 24–72 h post-differentiation (Figure 5A-B), in the form of radial migration of cells arising from the centre of the neurospheres and subsequent formation of neurite networks (arrowheads in Figure 5B). Scanning electron microscopy analyses (Figure 5C-D) confirmed extended neuritogenesis at day 20 of differentiation, newly formed neurites being often embedded by non-degraded nECM (arrows in Figure 5D). This “embedding effect” was also appreciated in Z-stack confocal views of the samples (see below). These data suggested a mechanical and trophic effect of the nECM on neuronal differentiation. On the other hand, SVZ cells started to show picnotic nuclei characteristic of cells entering apoptosis at 35 DIV (arrows in Figure 5E), a phenomenon that was not observed in nECM-based cultures.
To quantify the differentiation of SVZ progenitors on both PLL- and nECM-based substrates, the numbers of cells positive for MAP2 and TH were counted. At early time points (day 10) the percentage of dopaminergic neurons was not significantly different (p > 0.05) on both substrates (54.2 ± 10.3 vs 65.1 ± 15.8% MAP2+TH+ cells on PLL and nECM, respectively; Figure 6D-F). In contrast, a significant increase of dopaminergic neurons (84.0 ± 6.3% MAP2+TH+ neurons on nECM as compared to none on PLL) was found after 1 month in culture (Figure 6D-F).
In summary, the fact that SVZ progenitor cells maintained their multipotentiality for a week and thereafter differentiated towards dopaminergic neuron lineage for up to 35 days allowed us to temporarily map the expression of several proteins related to neuronal development, in what might be viewed as a relevant in vitro system for the assessment of unanswered questions related to neural development.
We have extended our previous observations on the ability of nECM to support in vitro neuronal differentiation, with the following achievements (i) we have recreated complex neuronal networks of embryonic rat hippocampal cells, and (ii) we achieved high levels of dopaminergic differentiation of SVZ progenitor cells. Furthermore, we showed proof-of-concept of the usefulness of this system to test the effect of added factors (as incorporated into the matrix) on neuronal behaviour. The biocompatible substrate enriched with nECM components represents a valid method to study neuronal development by using cells at different stages of commitment. Neural ECM-coated substrates increased culture maintenance window and neuronal differentiation efficiency as compared to cationic polymers such as PLL.
All experiments were carried out in accordance with the guidelines established by the National Council on Animal Care and were approved by the local Animal Care Committee of the Universidad Autónoma of Madrid (UAM, Madrid, Spain) or the University of Basque Country (Spain) Animal Ethics committee, as relevant, following European Communities Council Directive of 22 September 2010 (2010/63/EU). Every possible effort was made to minimize animal suffering and the number of animals used.
Isolation and culture of rat hippocampal cells
Pregnant Sprague–Dawley rats (Charles River Laboratories International, Inc., Wilmington, USA) were sacrificed by CO2 inhalation and 18-day embryos (E18) were immediately removed by caesarean section. Hippocampi were dissected rapidly by using a Leica CLX 150× stereomicroscope (Leica Microsystems, Barcelona, Spain) under sterile conditions in cold (4°C) PBS. The tissue was digested with 0.5 mg/ml papain (Sigma-Aldrich, Madrid, Spain) and 0.25 mg/ml DNAase (Sigma-Aldrich, Madrid, Spain). The enzymes were dissolved in a Ca2+- and Mg2+-free PBS solution containing 1 mg/ml BSA (Sigma-Aldrich, Madrid, Spain) and 10 mM glucose (Sigma-Aldrich, Madrid, Spain) at 37°C for 20 min. The papain solution was replaced with 5 ml Neurobasal™ A Medium (Life Technologies Corporation, Paisley, UK) supplemented with 2% B27 Supplement (Life Technologies Corporation, Paisley, UK), 1% L-glutamine solution 200 mM (Sigma-Aldrich, Madrid, Spain) and 1% penicillin/streptomycin solution (Sigma-Aldrich, Madrid, Spain). The digested tissue was gently triturated by suction using a glass pipette flamed on the tip to avoid cellular damage. The cell suspension was centrifuged for 4 min at 1,200 rpm. The supernatant was removed and the cells resuspended in 5 ml of medium and seeded on both PLL- and nECM-coated multiwell plates in the same maintenance medium at a density of 120,000 cells/ml. Cells were maintained in these conditions in a 5% CO2 incubator at 37°C.
Isolation and differentiation of rat SVZ progenitor cells
Neurosphere cultures were prepared from 4 to 7-day-old Sprague–Dawley rat pups (Charles River Laboratories International, Inc., Wilmington, USA). Subventricular zone (SVZ) tissue from 2–3 rat brains were isolated and minced with a McIllwain tissue chopper (H. Saur Laborbedarf, Reutingen, Germany) and digested for 10 min at 37°C in 5 ml of 0.25% trypsin/EDTA solution (Sigma-Aldrich, Madrid, Spain). Digestion was stopped by adding an equal volume of trypsin inhibitor (Sigma-Aldrich, Madrid, Spain) and 0.01% DNAse I for 5 min at room temperature. Cell suspension was centrifuged for 10 min at 600 g and the pellet was mechanically dissociated 25 times in NeuroCult medium (Stem Cell Inc., Grenoble, France) using a glass Pasteur pipette and 20 times using 1 ml pipette tips. Non-dissociated cells were decanted and the single cell suspension was counted using the Neubauer method. Cells were seeded in proliferation medium: NeuroCult with 10% neural stem cell factors (both from Stem Cell Inc., Grenoble, France), 1% L-glutamine solution 200 mM (Sigma-Aldrich, Madrid, Spain), 1% penicillin-streptomycin solution (Sigma-Aldrich, Madrid, Spain), 20 ng/ml EGF and 10 ng/ml FGF2 (both from Promega, Madrid, Spain), at a density of 10,000 cells/cm2 and cultivated in suspension for 7 days at 37°C with 5% CO2. EGF and FGF2 were added fresh every 2–3 days.
The differentiation of neurospheres on 12 mm-diameter PLL- and nECM-coated coverslips (10 neurospheres/coverslip) was carried out in Neurobasal™ A Medium supplemented with 2% B27 Supplement and 1% N2 Supplement (all from Life Technologies Corporation, Paisley, UK), 10% FBS (LGC Standards S.L.U., Barcelona, Spain), 1% L-glutamine solution 200 mM and 1% penicillin-streptomycin solution (both from Sigma-Aldrich, Madrid, Spain). Every two days, half of the medium was replaced and supplemented gradually and at different time periods up to 35 days with 50 ng/ml rat recombinant nerve growth factor β (NGFβ) (Sigma-Aldrich, Madrid, Spain), 50 ng/ml human recombinant brain derived neurotrophic factor (BDNF) (Sigma-Aldrich, Madrid, Spain), 200 ng/ml human recombinant Sonic hedgehog (Shh) (R&D, Minneapolis, USA) and 200 ng/ml mouse recombinant fibroblastic growth factor 8b (FGF8b) (R&D, Minneapolis, USA).
Development of neural extracellular matrix (nECM)-coated subtrates
Neural extracellular matrix (nECM)-coated substrates were developed as described . Briefly, 12 mm-diameter coverslips were incubated with a solution of Cultrex® basement membrane extract (BME) without phenol red (8.32 mg/ml; Trevigen, Gaithersburg, USA; it is a soluble form of basement membrane purified from Engelbreth-Holm-Swarm tumor; the major components of this BME include laminin, collagen IV, entactin, and heparin sulfate proteoglycan), hyaluronan of low molecular weight (31 kDa, HLMW) (7.5 mg/ml; R&D, Minneapolis, USA), Netrin-G1a and Netrin-4 (25 μg/ml; R&D, Minneapolis, USA) in phosphate-buffered saline (PBS) (Life Technologies Corporation, Paisley, UK). For some experiments, chondroitin 4-sulfate glycosaminoglycan (Sigma-Aldrich, Madrid, Spain) was added to the mix together with hyaluronic acid. In order to polymerize and adsorb the matrix on the exposed glass surface they were kept for 24 h at 37°C in sterile conditions. Finally, excess PBS was eliminated and coverslips left to dry up for 1 h under laminar flow and UV light. As a control, PLL-coated coverslips were prepared by incubation of 12 mm-diameter coverslips with a 0.01% solution of poly-L-lysine (Sigma-Aldrich, Madrid, Spain) in distilled water, followed by PBS washing and air drying under laminar flow for 1 h.
Immunofluorescence and confocal microscopy analyses
Differentiating cells were washed with PBS and fixed in 4% paraformaldehyde solution (Electron Microscopy Sciences, Hatfield, UK) for 20 min at room temperature. Cells were further washed with PBS and permeabilized for 1 hour in 0.3% Triton® X-100 (Sigma-Aldrich, Madrid, Spain) in PBS (PBS-T) and 5% normal donkey serum (Sigma-Aldrich, Madrid, Spain). After that, cells were incubated with the appropriate primary antibody diluted in PBS-T for 2 h at room temperature. Primary antibodies used were anti-Sox2 rabbit polyclonal (used at 1:1,000 dilution, Millipore #AB5603, Billerica, USA), anti-nestin mouse monoclonal IgG1 (1:200, Millipore #MAB353, Billerica, USA), anti-NeuN mouse monoclonal IgG1 (1:200, Millipore #MAB377, Billerica, USA), anti-GFAP mouse monoclonal IgG1 (1: 200; Dako, #M0761, Denmark), anti-MAP2 mouse monoclonal IgG1 (1:200; Millipore #MAB3418, Billerica, USA) and anti-tyrosine hydroxilase rabbit polyclonal (TH; 1:1,000; Millipore #AB152, Billerica, USA) antibodies. After 3 washes (5 min each) fixed cells were incubated with the appropriate secondary antibody diluted in PBS-T for 1 h at room temperature. Secondary antibodies used were donkey anti-mouse Alexa Fluor® 488 (1:1,000) and donkey anti-rabbit Alexa Fluor® 546 (1:1,000) (both from Life Technologies Corporation, Paisley, UK). Prior to embedding in Mowiol® mounting medium (Sigma-Aldrich, Madrid, Spain), cells were counterstained with 10 μg/ml Hoechst 33258 (Sigma-Aldrich, Madrid, Spain) for 5 min and washed with distilled water. Fluorescence images were obtained by using a Nikon Eclipse E600 FN microscope (objectives 10× and 20×) coupled to Nikon Digital Sight and analysed with Nikon NIS-Elements. Confocal images were generated with a Zeiss LSM 510 microscope coupled to Zeiss Axion camera and analyzed with Zeiss ZEN image analysis software (2008; SP1.1; Carl Zeiss MicroImaging, S.L., Barcelona, Spain). Z stack was analyzed with the plug in 3D viewer of Image J software. In both cases, at least ten different fields out of three independent experiments (n = 3) were quantified.
Quantification of cell viability with the MTT assay
Cell viability was assessed by quantitative colorimetric assay with (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), as previously described . Briefly, a final concentration of 0.5 mg/ml MTT labelling reagent (Sigma-Aldrich, Madrid, Spain) in Neurobasal™ A Medium was added to each well containing embryonic hippocampal cells cultured for 8 days on control nECM- and chondroitin 4-sulfate containing nECM-coated coverslips, and incubated for 2 h. After that, colorimetric determination of MTT reduction was measured at 540 nm with a Kontron Instruments UVIKON 922 spectrophotometer. These experiments were repeated after treatment of embryonic hippocampal cells with several cytotoxic agents such as glutamate, oligomycin-rotenone (O/R) and amyloid beta 25:35 (Aβ) (all from Sigma-Aldrich, Madrid, Spain) for 24 h. These experiments were performed with cells from day 7 to day 8 in culture. For statistical analyses, cells in each nECM-coated well in absence of toxic were taken as 100% of viability.
Scanning electron microscopy analyses
For Scanning Electron Microscopy (SEM) analyses, substrates with cells were fixed with Vitrosec® 70 (Panreac Química S.A.U., Barcelona, Spain) for 20 min and stained with May-Grünwald’s eosin methylene blue solution (Merck, Madrid, Spain). Samples were dried and mounted onto aluminium disks and coated with gold-argon. Imaging was performed on a JEOL JSM-5910-LV scanning electron microscope using a 20 kV acceleration voltage. Samples were analyzed by an INCA-300 energy dispersed system (EDS) coupled to the microscope.
Data are expressed as the mean ± SD of the number of independent experiments (n). Statistical significance of the results was assessed by using GraphPad Prism software (version 5.01 for Windows). Two-way analysis of variance with subsequent pairwise multiple comparison procedures (Bonferroni’s posttests) was used to assess statistical significance between means. The statistical significance (*) was established at P-values <0.05 (** P < 0.01; *** P < 0.001).
Neural-like extracellular matrix
Neural stem cells
Hyaluronan of low molecular weight
This work was financed by grants from the Department of Industry and Commerce of the Basque Government (PC200829A-NEUROPATX, Spain) and Ministerio de Ciencia e Innovación (PI10/02871) from Spain. P.G.-P. was first supported by a fellowship from the “Iñaki Goenaga” Technology Centres Foundation (FCT-IG) (Technology Development Grants for Young Scientists, Spain) and then by a postdoctoral contract funded by Ministerio de Ciencia e Innovación (MICINN, Spain) [INNPACTO program (IPT-300000-2010-17). AGG and MM were supported by: (1) SAF 2010–21795, Ministerio de Economía y Competitividad (Spain); (2) RENEVAS-RETICS-RD06/0026, Instituto de Salud Carlos III (Spain); (3) CABICYC, UAM/Bioibérica (Spain). F.C. was supported by a project grant funded by Ministerio de Ciencia e Innovación (MICINN, Spain) (SAF2009-13463). A.I. was supported by the “Programa I3SNS” (CES09/015) from Instituto de Salud Carlos III and by Osakidetza-Servicio Vasco de Salud (Spain). We thank the continued support of Fundación Teófilo Hernando of Madrid (Spain).
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