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In Vivo Bioluminescence Imaging for Viable Human Neural Stem Cells Incorporated Within in Situ Gelatin Hydrogels

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In Vivo Bioluminescence Imaging for Viable Human Neural Stem Cells Incorporated Within in Situ Gelatin Hydrogels Hwang et al. EJNMMI Research 2014, 4:61 http://www.ejnmmires.com/content/4/1/61 ORIGINAL RESEARCH Open Access In vivo bioluminescence imaging for viable human neural stem cells incorporated within in situ gelatin hydrogels Do Won Hwang1,2, Kyung Min Park3, Hye-kyung Shim1, Yeona Jin1, Hyun Jeong Oh1,2, So Won Oh1, Song Lee1, Hyewon Youn1,4,5, Yoon Ki Joung3, Hong J Lee6, Seung U Kim6,7, Ki Dong Park3* and Dong Soo Lee1,2* Abstract Background: Three-dimensional (3D) hydrogel-based stem cell therapies contribute to enhanced therapeutic efficacy in treating diseases, and determining the optimal mechanical strength of the hydrogel in vivo is important for therapeutic success. We evaluated the proliferation of human neural stem cells incorporated within in situ-forming hydrogels and compared the effect of hydrogels with different elastic properties in cell/hydrogel-xenografted mice. Methods: The gelatin-polyethylene glycol-tyramine (GPT) hydrogel was fabricated through enzyme-mediated cross-linking reaction using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). Results: The F3-effluc encapsulated within a soft 1,800 pascal (Pa) hydrogel and stiff 5,800 Pa hydrogel proliferated vigorously in a 24-well plate until day 8. In vitro and in vivo kinetics of luciferase activity showed a slow time-to-peak after D-luciferin administration in the stiff hydrogel. When in vivo proliferation of F3-effluc was observed up to day 21 in both the hydrogel group and cell-only group, F3-effluc within the soft hydrogel proliferated more vigorously, compared to the cells within the stiff hydrogel. Ki-67-specific immunostaining revealed highly proliferative F3-effluc with compactly distributed cell population inside the 1,800 Pa or 5,800 Pa hydrogel. Conclusions: We examined the in vivo effectiveness of different elastic types of hydrogels encapsulating viable neural stem cells by successfully monitoring the proliferation of implanted stem cells incorporated within a 3D hydrogel scaffold. Keywords: Gelatin-based hydrogel; Matrix elasticity; Human neural stem cell; In vivo bioluminescence imaging; Optical kinetics Background the injured and inflamed tissues. This remains a critical Rapid advances in stem cell-based therapy have opened limitation for successful cell therapy. To overcome this up the possibility of stem cell use to reconstitute injured challenge, a variety of biomaterials such as microfiber- tissues for the functional improvement in the clinic. Par- type or gel-type scaffolds have been developed to support ticularly, neural stem cells, capable of being differenti- survival and proliferation of implanted stem cells [5-11]. ated into functional neurons, could become a good cell Among the many scaffolds currently available, hydrogels, source for the treatment of neurodegenerative diseases capable of imbibing large amounts of water and possessing [1-4]. In spite of this progress, studies concerning stem suitable physicochemical properties, are known to exhibit cell therapy have shown poor survival rates for the im- the best biocompatibility and biodegradability in vivo.The planted stem cells, owing to the necrotic environment of hydrogel materials can be fabricated by various chemical and physical reactions, and their physiochemical proper- * Correspondence: [email protected]; [email protected] ties, such as gelation, mechanical properties, and degrad- 3 Department of Molecular Science and Technology, Ajou University, ation time, could be easily controlled [12,13]. In addition, 5 Woncheon, YeongtongSuwon 443-749, Republic of Korea 1Department of Nuclear Medicine, Seoul National University College of the hydrogels can encapsulate therapeutic drugs and cells Medicine, 28 Yongon-Dong, Jongno-Gu, Seoul 110-744, Republic of Korea for the treatment of targeted diseases as they are formed Full list of author information is available at the end of the article © 2014 Hwang et al.; licensee Springer. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Hwang et al. EJNMMI Research 2014, 4:61 Page 2 of 11 http://www.ejnmmires.com/content/4/1/61 in mild physiological conditions. These characteristics Methods facilitated the use of hydrogels in therapeutic cell implants Synthesis of GPT conjugate or as therapeutic delivery vehicles [14-16]. Hydrogels, In our previous report, the GPT hydrogel was developed which are cross-linked hydrophilic polymers, can serve as as an injectable material with excellent biocompatibility a bio-artificial niche not only for promoting survival of the and bioactivity for tissue regeneration and drug delivery implanted cells, but also for protection of the cells from [22]. The GPT conjugate was synthesized by coupling tyr- the damaged tissue environment in vivo, by providing amine (TA)-conjugated polyethylene glycol (PNC-PEG-TA) mechanical framework and three-dimensional super- and gelatin. Briefly, the hydroxyl groups of polyethylene gly- porous structures. The matrix hydrogel elasticity could col (PEG) reacted with p-nitrophenyl chloroformate (PNC) be adjusted to match the stiffness of real tissues (brain: to activate the terminal groups (PNC-PEG-PNC), and then 0.1 to 1 kilopascal [kPa]; muscle: 8 to 17 kPa; collage- the PNC-PEG-PNC reacted with TA and gelatin to give the nous bone: >34 kPa) and optimized to induce specific GPT polymer. The chemical structure and degree of substi- cell fates from the implanted mesenchymal stem cells tution (DS) of TA were characterized by 1HNMRandUV 1 [17-19]. Furthermore, studies have shown that hydrogel- measurements ( HNMR(D2O): δ 4.8 (m, the proton encapsulated cells were functionally effective in several of anomeric carbon of gelatin), δ 0.8 to 4.6 (m, alkyl disease models [11,20,21]. Despite this effectiveness of proton of gelatin), δ 3.5 to 3.8 (m, -CH2-CH2 of PEG the biomimetic hydrogel, the characteristics of different ethylene), and δ 6.8 and 7.1 (m, aromatic protons of in vivo hydrogels are not understood for their real in vivo TA)). The TA content in the polymer was determined by behavior of hydrogel-encapsulated cells. An in vivo im- UV-vis spectroscopy measurements at a wavelength of aging technique that tracks the survival of implanted stem 275 nm. Utilizing a tyramine standard curve, we found cells within the hydrogel will help evaluate the in vivo that the TA content in the GPT conjugate was 110 μmol/g efficacy of different hydrogel matrix types. of GPT polymer. Gelatin (type A from porcine skin, >300 The gelatin-polyethylene glycol-tyramine (GPT) hydrogel, Bloom), PEG (MW 4,000), HRP (250 to 330 units/mg recently developed in our group, is an in situ cross-linkable solid), aqueous hydrogen peroxide (H2O2; 30% [w/w]), hydrogel that exhibits rapid gel formation induced by the 4-dimethylamino pyridine (DMAP), and PNC were pur- cross-linking reaction of horseradish peroxidase (HRP) with chased from Sigma-Aldrich (St. Louis, MO, USA). TA hydrogen peroxide (H2O2) [22]. This enzyme-mediated was obtained from Acros Organics (Geel, Belgium). type of hydrogel possesses significant advantages of Triethylamine (TEA) was supplied by Kanto Chemical excellent biocompatibility and controllable mechanical Corp. (Tokyo, Japan). Aluminum oxide (Al2O3) was pur- strength. In addition, because this hydrogel is compat- chased from Strem Chemicals (Newburyport, MA, USA). ible with an injection system that can easily be applied Other chemical reagents and solvents were used without in vivo, with minimal surgical invasiveness, it has the further purification. potential for use in tissue engineering-based regenera- tive therapy [22,23]. Preparation of the hydrogels and measurement of the Bioluminescence light is emitted from the catalytic elastic modulus (G′) reaction of luciferase enzyme with its substrate, and The GPT hydrogels (200 μL) were prepared through in vivo administration of D-luciferin can be used to gen- enzyme-mediated reaction using HRP and H2O2. One- erate bioluminescence in implanted luciferase-expressing hundred microliters of GPT polymer solution (3% w/w) stem cells encapsulated within the hydrogel in small an- dissolved in HRP stock solution and another 100 μLdis- imals. The permeability of D-luciferin within the hydro- solved in H2O2 solution (0.0038% to 0.0075% w/w) were gel may differ according to its mechanical strength. mixed to form the hydrogels. All solutions were dissolved Therefore, examining the in vivo kinetics of the lucifer- in 0.01 M phosphate-buffered saline (PBS; pH 7.4). ase activity in the living mouse bearing the hydrogel- The elastic modulus (G′) was measured with an Ad- encapsulated stem cells after D-luciferin administration vanced Rheometer GEM-150-050 (Bohlin Instruments, is necessary to acquire the optimal bioluminescence sig- East Brunswick, NJ, USA) using the parallel plate (20-mm nal in implanted stem cells within hydrogels of different diameter) configuration at 37°C in oscillatory mode. The elasticity. GPT polymer was dissolved both in HRP solution In this study, we investigated the survival and prolifer- (0.0005 mg/mL of stock solution) and in solutions con- ation of injectable hydrogel-encapsulated stem cells by taining different concentrations of H2O2 (0.0038% to non-invasively monitoring human neural stem cells car- 0.0075% w/w of stock solution). Two-hundred micro- rying the highly sensitive luciferase gene. Based on this liter polymer solutions containing HRP and H2O2 were in vivo imaging strategy, cell survival and proliferation in rapidly mixed on the bottom plate of the instrument, soft and stiff hydrogels were evaluated in nude mice with and the upper plate was immediately lowered down to analysis of in vivo kinetics of the luciferase substrate. ameasuringgapsizeof1mm.Afrequencyof0.1Hz Hwang et al. EJNMMI Research 2014, 4:61 Page 3 of 11 http://www.ejnmmires.com/content/4/1/61 (single frequency) and a strain of 0.1% (strain control) after D-luciferin administration was examined in vitro were applied for the analysis to maintain a linear visco- using a microplate luminometer for a period of 95 min elastic response.
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