Research Article

In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles

Aim: Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cells into MN precursors involves factors and their repeated administration. We investigated if delivery of factors loaded into mesoporous nanoparticles could be effective for stem cell differentiation in vitro. Materials & methods: We used a mouse embryonic stem cell line expressing green fluorescent protein under the promoter for the MN-specific gene Hb9 to visualize the level of MN differentiation. The differentiation of stem cells was evaluated by expression of MN-specific transcription factors monitored by quantitative real-time PCR reactions and immunocytochemistry. Results: Mesoporous nanoparticles have strong affiliation to the embryoid bodies, penetrate inside the embryoid bodies and come in contact with differentiating cells. Conclusion: Repeated administration of soluble factors into a culture medium can be avoided due to a sustained release effect using mesoporous silica. Original submitted 28 August 2013; Revised submitted 23 January 2014

Keywords: cell transplant • differentiation • mesoporous material • nanomedicine • stem cell

Mesoporous silica nanoparticles possessing ordered porosity in the mesoscale (2–50 nm) are being extensively researched in nanomedicine for diagnostics, controlled drug-release areas and pharmaceutical formulation [1–3]. The ability of these nontoxic and highly porous amorphous silica particles to release poorly soluble pharmaceutical compounds in their noncrystalline state from within their pores significantly enhances their aqueous solubility and their bioavailability [4,5]. Several reliable strategies have emerged to tailor the release kinetics of pharmaceutical drugs, DNA, siRNA, nutraceuticals and growth factors from the internal pore space of mesoporous materials. These include structural variations in the pore diameter, electrostatic or covalent interactions between the wall and the drug, functionalization of the internal pore space, particle PEGylation, and external functionalization of the pore entrances [6]. A change in pore structure alone, from 2D to 3D pore connectivity, has been shown to

10.2217/NNM.14.23 © 2014 Future Medicine Ltd

increase the pharmacokinetic properties of model drugs from the internal pore space by several orders of magnitude [7]. The desire to tailor release properties via an external (e.g.,  optical response or magnetic field) or internal (e.g., gastric changes in pH) stimuli has led to several interesting pore gatekeeping strategies. Intelligent drug-delivery vehicles based on mesoporous silica particles that respond in situ to changes in blood plasma composition of certain disease markers have already been achieved and represent the next wave of drug-delivery technologies. Lin and colleagues developed glucose-triggered release of both gluconic acid-modified insulin and cAMP from mesoporous particles by immobilizing gluconic acid-modified insulin on the exterior surface of the particles. The immobilized insulin served as a gatekeeper to encapsulate cAMP inside the mesopores [8]. With a similar goal, Zhao et al. have achieved triggered release of insulin based on sequential coatings of enzymatic layers crosslinked with

Nanomedicine (Lond.) (2014) 9(16), 2457–2466

Alfonso E Garcia-Bennett*,1,‡, Niclas König2,‡, Ninnie Abrahamsson2, Mariya Kozhevnikova2, Chunfang Zhou3, Carl Trolle2, Stanislava Pankratova4, Vladimir Berezin4 & Elena N Kozlova*,2 Department of Materials & Environmental Chemistry, Arrhenius Laboratory, Stockholm University, SE-106 91, Stockholm, Sweden 2 Department of Neuroscience, Uppsala University Biomedical Center, Box 593, SE-751 24, Uppsala, Sweden 3 Nanologica AB, Drottning Kristinas väg, 62, SE-114 28, Stockholm, Sweden 4 Laboratory of Neural Plasticity, Department of Neuroscience & Pharmacology, Panum Institute, Copenhagen University, Copenhagen, Denmark *Authors for correspondence: [email protected] [email protected] ‡ These authors contributed equally 1

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Research Article  Garcia-Bennett, König, Abrahamsson et al. glutaraldehyde, which were shown to act as a valve to control the release of insulin in response to mesoporous silica particles [9]. A potential, and hitherto unexplored, application for mesoporous particles is their use as a delivery vehicle in vitro for the long-term release of differentiation factors in a controlled, slow-release manner. Stem cell studies, particularly with human stem cells, require repeated delivery of differentiation factors during many weeks of culture to guide the differentiation of embryonic stem cells (ESCs) towards specific neural phenotypes [10]. As a result the concentration of the relevant factors in the culture medium varies, which can negatively affect the differentiating process and lead to bothersome variability in the differentiation results. Retinoic acid (RA) and Shh are morphogens of fundamental and widespread importance during the development of the nervous system, both as regulators of patterning as well as for participating in directing stem cells in the embryonic nervous system towards neuronal progenitors [11,12]. RA and Shh (or its agonist purmorphamine; PUR) are the basis for in vitro protocols for generating motor neurons (MNs) progenitors from mouse (mESCs) [13] and human [14] ESCs. Here, we use a generation of MNs from mESCs as a model to explore the efficacy of continuous and long-term mesoporous silica-mediated release of morphogens compared with the conventional procedure of their repeated administration. Materials & methods Mesoporous silica

The synthesis of cubic AMS-6 mesoporous silica has been reported previously and can be found in detail in [7], and references within. The mesoporous particles (AMS-6: 200 nm diameter particle with a 5.8 nm pore size) were loaded with either RA (AMS-6–RA) or PUR (AMS-6–PUR). The loading of the factors was verified by thermogravimetric analysis, showing loading capacities of 18.5 and 6.5 wt% for RA and PUR, respectively. After loading, nitrogen adsorption isotherms demonstrated that the mesoporous material pore volume and textural properties decreased compared with unloaded particles, implying the successful incorporation of the factors within the pores (see Supplementary Material online at www. futuremedicine.com/doi/suppl/10.2217/NNM.14.23). To investigate the in vitro relationship between nanoparticles and ESCs the AMS-6 particles were labeled with rhodamine and added, together with AMS-6–RA particles, to the culture medium. Briefly, surfactant-extracted AMS-6 particles (1 g) were refluxed for 24 h in ethanol that contained the desired amount of tetramethylrhodamine-5-(and 6)-isothiocyanate (TRITC; 5 mg; Sigma-Aldrich, MO, USA) at pH 11

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(obtained by the addition of NaOH). The sample was filtered and dried prior to TRITC conjugation. The remaining pink solid was then filtered, washed with distilled water (50 ml) followed by ethanol (250 ml) to remove unreacted TRITC, and left to dry at room temperature. Cell culture

We used an mESC line carrying a transgenic reporter gene in which expression of green fluorescent protein (GFP) is driven by the promoter element from the Hb9 gene (Hb9::GFP) [13]. The mESCs were cultured according to a previously published protocol [13]. ESC colonies were partially dissociated after 2 days to form embryoid bodies (EBs) and supplemented with 0.1 µM all-trans RA (Sigma-Aldrich) and 0.5 µM of PUR (Calbiochem®; MerckMilipore, Damstadt, Germany) in free form or delivered with AMS-6. EBs were cultured for an additional 3 days when Hb9::GFP-expressing cells appeared. To investigate the interrelations between AMS particles and EBs in vitro, rhodamine-labeled particles were added with AMS-6–RA or AMS-6–PUR in a ratio of 1:9 on day 1 in culture. The collected EBs were fixed with 4% paraformaldehyde, and embedded into Tissue-Tek® (Sakura Finetek, Alphen aan den Rijn, The Netherlands), and 6-µm cryosections were prepared for subsequent ana­lysis by confocal microscopy. In addition, anti-b-catenin antibodies were used to visualize the membranes of the cells in EBs (Abcam®, Cambridge, UK). Quantitative real-time PCR

Total RNA from four culture experiments was isolated using the RNeasy® Mini Kit (Qiagen, Hilden, Germany). For the ana­lysis of the quantitative PCR data, CFX Manager™ software (Bio-Rad Laboratories Inc., CA, USA) was used, in which the relative expression of the gene of interest was calculated on the basis of the DDCq method (Livak method). The CFX Manager Software, using the DDCq method, sets the expression level of the control sample (day 0) to one automatically. The plots were made using the Prism 5 Software (GraphPad, CA, USA). The GAPDH gene was used as a housekeeping gene. The High-Capacity cDNA Reverse-Transcription Kit (Applied Biosystems Inc., MA, USA), including MultiScribe™ Reverse Transcriptase (Applied Biosystems Inc.) and random primers were used for cDNA synthesis. Quantitative real-time PCR was performed using 2 × SYBR® Green Master Mix (Invitrogen, CA, USA) in a 25-µl reaction mixture in the MyiQ™ real-time PCR detection system (Bio-Rad Laboratories Inc.). The reaction was performed under

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In vitro generation of MN precursors from mouse embryonic stem cells using mesoporous nanoparticles 

the following conditions: 95°C hold for 5 min, 40 cycles of 95°C for 15 s, 55–60°C (depending on primer pairs) for 12–30 s, and 72°C for 12 s. The primers (Table 1) were designed using Beacon Designer software (PREMIER Biosoft International, CA, USA). Immunocytochemistry & evaluation of immunofluorescence in EBs

Sections from ten culture experiments were treated according to a previously published protocol [15]. The primary and secondary antibodies used in this study are presented in Table 2. To evaluate the percentage of immunofluorescent cells in EBs treated with RA and PUR delivered in free from or with AMS, the EBs from ten experiments from each condition were collected and 6 µm sections were prepared and stained with Hoechst nuclear staining, and images from each experiment were taken. The percentage of GFP+ cells with blue nuclei in relation to all blue nuclei in the EB image was calculated for seven randomly chosen EBs from each experiment. Values are expressed as the mean ± the standard error of the mean. Student’s unpaired t-test was used. For all comparisons, p 

In vitro generation of motor neuron precursors from mouse embryonic stem cells using mesoporous nanoparticles.

Stem cell-derived motor neurons (MNs) are utilized to develop replacement strategies for spinal cord disorders. Differentiation of embryonic stem cell...
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