以氧化鐵奈米粒子標記人類間質幹細胞誘導成為類神經細胞的追蹤表現及應用

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(1)國立臺灣師範大學生命科學系博士論文. 以氧化鐵奈米粒子標記人類間質幹細胞誘導成為類神經細胞的追蹤表現及應用 Characterization of an iron oxide nanoparticle labelling and MRI-based protocol for inducing human mesenchymal stem cells into neural-like cells. 研究生：盧珍妏 (Chen-Wen, Lu) 指導教授：吳忠信博士 (Chung-Hsin Wu, Ph.D.). 中華民國 106 年 07 月 17 日.

(2) TABLE OF CONTENTS TABLE OF CONTENTS. I. LIST OF FIGURES. IV. 中文摘要. V. ABSTRACT. VI. CHAPTER 1 INTRODUCTION. 1. 1.1 HMSCs. 2. 1.2 Iron oxide nanoparticles (IONs). 3. 1.3 Research aims. 4. CHAPTER 2 MATERIALS AND METHODS 2.1 Cell culture. 5 6. (1) Human mesenchymal stem cell culture (2) Neurogenic differentiation of human mesenchymal stem cells. 7. (3) ION labelling. 2.2 Morphological analysis. 7. (1) Transmission electron microscopy (2) Co-staining with Prussian Blue and phosphotungstic acid. 8. haematoxylin (PTAH) 2.3 Reverse transcription polymerase chain reaction (RT-PCR). 8. 2.4 Western blotting. 9. 2.5 Immunofluorescence staining. 10. 2.6 Flow cytometry analysis of neural markers. 11. -I-.

(3) 2.7 Electrophysiological recording. 11. 2.8 Flow cytometry detection of ION particle uptake. 12. 2.9 Magnetic resonance imaging (MRI). 12. 2.10 Intracellular iron content determination. 13. 2.11 Viability assay. 13. 2.12 Reactive oxygen species measurements. 14. 2.13 Mitochondria membrane potential measurements. 14. 2.14 Statistical analysis. 15. CHAPTER 3 RESULTS. 16. 3.1 Differentiated human MSCs exhibited neural-like morphology 17 and neuron markers: Directly labelling hMSCs and NCs with ION 3.2 Electrophysiological function. 18. 3.3 In vitro determination of ION uptake by MRI, inductively 19 coupled plasma mass spectrometry (ICP-MS) and flow cytometry 20. 3.4 Cell behavior CHAPTER 4 DISCUSSION. 21. 4.1 Cell behavior. 22. 4.2 Different sizes of supraparamagnetic ION. 23. 4.3 NPs coating. 26 - II -.

(4) 4.4 Iron Content. 29. 4.5 Neural induction medium (NIM). 29. 4.6 NCs. 30. 4.7 Neural Protein expression. 31. 4.8 Spontaneous firing frequency. 32. CHAPTER 5 CONCLUSION. 34. CHAPTER 6 REFERENCES. 36. CHAPTER 7 FIGURES. 57. CHAPTER 8 Tables. 69. APPENDIX 1: CV of Chen-Wen, Lu. i. APPENDIX 2: Copy of Published Sci Papers. ii. - III -.

(5) LIST OF FIGURES and TABLES FIGURES. 57. Figure 1. Comparison of hMSC differentiation capacity into NCs with or without (w/o) ION. 58. Figure 2. TEM images of ION (Resovist, ferucarbotran). 60. Figure 3. Characterization of neural differentiation markers in hMSCs treated with or without neural induction medium after ION labelling. 61. Figure 4. Action potentials of hMSCs, NCs with or without ION labelling. 63. Figure 5. Quantification of iron content after labelling with or without ION before and after induction of neural-like cell differentiation. 65. Figure 6. Measuring cell behaviour using three different assays. 67. Table. 69. 1. Primer sequences used for RT-PCR analysis. - IV -.

(6) 中文摘要. 本研究的目的是以開發氧化鐵奈米粒子(ION)標記人類間質幹細胞 (MSCs) 誘導體外分化成為類神經細胞 (NCs) 的應用以及在核磁共振成像（MRI）之追蹤表現。Ferucarbotran，一種臨床所使用的氧化鐵奈米粒子，此種陰性顯影劑可以在核磁共振成像下清楚的看見，因此被用來標記細胞內的追蹤觀察。本研究透過光學顯微鏡下發現體外培養的類神經細胞具有神經細胞的型態以及量測動作電位的功能表現。在光學顯微鏡下觀察到細胞呈現軸突樣的結構型態。這些類神經細胞比未分化的間質幹細胞表現較多頻率的動作電位。以氧化鐵奈米粒子標記對間質幹細胞的形態、功能和分化能力沒有影響。我們的結論發現，以體外誘導人類間質幹細胞 (MSCs) 分化成的類神經細胞 (NCs) 表現較多頻率的動作電位，或許這些體外誘導生成的類神經細胞可以用於替代損傷的神經元。. 關鍵詞：氧化鐵奈米粒子、核磁共振成像、間質幹細胞、類神經細胞、動作電位. -V-.

(7) ABSTRACT The aim of the current study was to develop an iron oxide nanoparticle (ION) labelling and magnetic resonance imaging (MRI)-based protocol to allow visualization of the differentiation process of mesenchymal stem cells (MSCs) into neural-like cells (NCs) in vitro. Ferucarbotran, a clinically available ION, which can be visualized under MRI, is used for tracking cells implanted in vivo. The NCs were verified morphologically and histologically by light microscopy, and their functions were verified by measuring their action potentials. Conformational conversion of axon-like structures was observed under light microscopy. These NCs exhibited frequent, active action potentials compared with cells that did not undergo neural differentiation. The labelling of ION had no influence on the morphological and functional differentiation capacity of the MSCs. We conclude that the MSCs that were differentiated into NCs exhibited in vitro activity potential firing and may be used to replace damaged neurons.. KEYWORDS: iron oxide nanoparticle (ION), magnetic resonance imaging (MRI), mesenchymal stem cells (MSCs), neural-like cells (NCs), action potential. - VI -.

(8) CHAPTER 1. INTRODUCTION. -1-.

(9) 1.1 HMSCs Mature neurons do not replicate, which limits their capacity for tissue repair in many conditions such as ischaemic stroke, spinal cord injury, traumatic brain injury, or neurodegenerative diseases1-4. Replacing damaged neurons via tissue engineering is theoretically possible in such conditions. Advanced cell imaging is required for administering stem cell therapy via tissue engineering techniques. Stem cell therapies can be potentially used to treat neurological diseases, either for replacing lost neurons, restoring neural circuits or as paracrine-mediated therapies5-7. Human mesenchymal stem cells (hMSCs) are multipotent cells that differentiate into bone, osteocytes, cartilage cells, adipocytes and neurons6-10. The differentiation potential of hMSCs into ectodermal cells11-16, such as astrocytes, neurons17-19 and oligodendrocytes20, has therapeutic potential for neurological diseases16,18,19. Although the replacement of the damaged neuron by tissue engineering is not well established, implantation of differentiated neurons is one of the most promising methods. However, monitoring the fate and migrating path of implanted neurons is difficult unless a powerfull imaging technique could be applied. Tracking transplanted cells in vivo provides direct real-time information on the cell migration, homing, division and/or differentiation, and survival of transplanted cells. Some researchers successfully implant iron oxide based nanoparticles into different kinds of cells and monitor the cell fate under magnetic resonance imaging2,4,21 and yields satisfactory result.. -2-.

(10) Magnetic resonance imaging (MRI) is an excellent tool for studying the fate of transplanted stem cells in vivo because it is non-invasive and inherently offers high spatial resolution, the absence of radiation and unlimited tissue penetration depth. In addition, successful monitoring and tracking of stem cells labelled with iron oxide nanoparticles (IONs) has been reported4.. 1.2 Iron oxide nanoparticles (IONs) Iron oxide nanoparticles have been widely used as clinical contrast agents in MRI for the detection of liver tumours22-28. ION can be internalized into neuron progenitor cells and visualized by MRI for up to 7 days29. Once ingested by macrophages or the reticuloendothelial system such as Kupffer cells, ION are metabolized, and the iron core is recycled into the tissue iron pool for the synthesis of haemoglobin. The remainder of the nanoparticle shell, which is primarily composed of sugar-related polymers, is excreted by the kidneys and making it perfect biodegradable nanoparticle.. The iron oxide core is paramagnetic, allowing it been detected under clinical MRI especially in liver tumor30-32. Owing to the absence of radiation and umlimited tissue penetration depth MRI has become the most popular imaging tool to detect brain disease such as ischemic stroke, dementia and other metabolic disease33-37. Our previous study on hMSCs, which were successfully labelled with ION, revealed no significant change to cellular behaviours, such as viability, mitochondrial membrane potential changes or differentiation capacity38.. -3-.

(11) 1.3 Research aims Our goal is to establish a platform to non-invasively imaging mesenchymal stem cells differentiated into neurons preserving neuronal morphologically and physiologically functional. These cells will be exposed to clinical available IONs, Ferucarbotran, before the cells have been differentiated, allowing is imaging capability by MRI36,37,39. The cell morphology, specific markers and function will be verified after differentiation and iron oxide uptake. The platform is hopefully being used to traffic implanted cells in living animals.. -4-.

(12) CHAPTER 2. MATRIALS & METHODS. -5-.

(13) 2. Materials and methods All experimental procedures for hMSCs culture were approved by the Committee on Biological Research of National Taiwan Normal University and implemented under the guidelines of the Committee.. 2.1 Cell culture (1) Human mesenchymal stem cell culture hMSCs that had been immortalized through the transfection of human telomerase reverse transcriptase (hTERT) with human papillomavirus E6 and E7 were a gift from Li-Horn40. The culture medium (CM) consisted of high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, BRL, Grand Island, NY, USA), supplemented with 10 % foetal bovine serum (FBS; HyClone, Logan, UT, USA), 4 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, NY, USA) (Gibco BRL, Grand Island, NY)19.. (2) Neurogenic differentiation of human mesenchymal stem cells HMSCs labelled with or without ION were plated at a density of 3 x104 cells/cm2 in 6-well culture plates. When the cells were confluent, neurogenic differentiation was initiated by NIM culture containing DMEM (Gibco BRL, Grand Island, NY) with 10 % FBS, 10 ng/mL basic-fibroblast growth factor (b-FGF), 10 ng/mL epidermal growth -6-.

(14) factor (EGF), 10 ng/mL brain-derived growth factor (BDGF, R&D Systems, Minneapolis, MN, USA), 1 ng/mL platelet-derived growth factor (PDGF, R&D Systems, Minneapolis, MN, USA), 0.5 mM all-trans retinoic acid (ATRA), 1X N1 medium supplement solution (Sigma-Aldrich Co., St. Louis, MO, USA), 2 mM valproic acid (Sigma-Aldrich Co.), and 10 µM forskolin and 1 M hydrocortisone ((Sigma-Aldrich Co.). The induction medium was changed every 2-3 days for 21 days.. (3) ION labelling For incubation with or without ION, ferucarbotran ION (Resovist®, Bayer Pharma AG, Berlin, Germany) were added to the culture medium at 100 µg Fe/mL. After 24 hours incubation at 37°C in 5 % CO2, the cells were further evaluated using Prussian Blue staining (Sigma-Aldrich Co.) and MRI. Examination of neural protein expression through flow cytometry, immunofluorescence, Western blotting and RT-PCR analysis were also conducted.. 2.2 Morphological analysis (1) Transmission electron microscopy We used permanent magnets to observe the concentration of iron oxide ions by placing 10 µg/ml and 100 µg/ml ION in a centrifuge tube. The intracellular 100 µg/ml ION uptake by cells was confirmed by transmission electron microscopy (TEM). 1x104 labelled or unlabelled cells were cultured in a plastic chamber slide (Lab-Tek, Nunc, Naperville, Il, USA) overnight. After washing with phosphate-buffered saline (PBS; Sigma-Aldrich Co.), the cells were fixed with Karnovsky’s fixation solution containing 2 -7-.

(15) % paraformaldehyde (Sigma-Aldrich Co.) with 2.5 % glutaraldehyde (Sigma-Aldrich Co.) in 0.2 M cacodylate (pH 7.4) (Sigma-Aldrich Co.) for 2 hours at 4°C, followed by incubation with 1 % osmium tetroxide (OsO4) buffer for 1.5 hours in the dark for post-fixation, rinsing, dehydration, and embedding.12 Ultra-thin slices were cut from the dried sections with a diamond knife and placed on the grids. Photographic images were taken using a TEM with a CCD camera (Hitachi H-7100; Hitachi, Ibaraki, Japan).. (2). Co-staining. with. Prussian. Blue. and. phosphotungstic. acid. haematoxylin (PTAH) To localize the intracellular ION, 1x105 hMSCs were exposed to 100 µg Fe/mL ION (Resovist, 45-60 nm) (Schering AG, Berlin, Germany) for 24 hours and then transferred to NIM and incubated for 2~3 weeks. The hMSCs were treated with a 1:1 mixture of 2 % potassium ferrocyanide (Prussian Blue) and 1 M hydrochloric acid for 5 minutes. Furthermore, the hMSCs and NCs were stained for astrocytes, fibroglia and myoglia using PTAH (Sigma-Aldrich Co.) for 20 minutes at room temperature. The cells were then washed twice and imaged using a Nikon TE2000-S inverted microscope.. 2.3 Reverse transcription polymerase chain reaction (RT-PCR) Total RNA from hMSCs and NCs was extracted with TriZol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Briefly, the cells were harvested after 21 days of incubation and lysed in supplied lysis buffer. Total RNA concentration was determined by measuring the optical density at 260 nm (OD260) using a spectrophotometer (DU800; Beckman Coulter, Fullerton, CA, USA). One microgram -8-.

(16) total RNA was used to generate cDNA using a SuperScript III First-Strand cDNA Synthesis System (Invitrogen, Carlsbad, CA, USA). Polymerase chain reaction (PCR) conditions and cycle numbers for a linear amplification range were determined and optimized. The primers used are shown in Supplement Table 1 (S1). The PCR amplification was carried out using PCR Tag Master Mix (Applied Biosystems, Foster City, CA, USA). The thermal profile for PCR was 96°C for 5 minutes, followed by 40 cycles of 96°C for 50 s, 59~63°C for 90 s, and 72°C for 90 s. The products were examined by electrophoresis in a 2 % agarose gel, stained with ethidium bromide and visualized under UV light. Actin was used as a housekeeping gene. The gene expression level was quantitated using the National Institutes of Health (NIH) ImageJ program (National Institutes of Health, USA). The expression level of housekeeping gene was defined as 1.0. The expression ratios of hMSC-derived neural specific genes to the housekeeping gene were determined41.. 2.4 Western blotting HMSCs and NCs lysates were prepared according to standard procedures. Protein content was quantified using a BCA protein assay kit (Pierce, Rockford, IL, USA). Ten micrograms total protein from each lysate was separated by 10 % sodium dodecyl sulphate–polyacrylamide. gel. electrophoresis. (SDS–PAGE). and. transferred. electrophoretically to a polyvinylidene fluoride (PVDF) transfer membrane (NEN Life Science Products, Boston, MA, USA). All membranes were blocked with 5 % nonfat dry milk in PBS for 1 hour at room temperature, then incubated with mouse anti-neuron specific enolase (NSE), anti-GFAP, anti-TH, anti-β-III tubulin (Tuj1) (1:1000; Millipore, Billerica, MA, USA) and anti-actin (1:2000; Millipore, Billerica, MA, USA). After washing. with. PBST,. the. membrane -9-. was. incubated. with. horseradish.

(17) peroxidase-conjugated secondary antibodies (human anti-mouse IgG, 1:10,000; Millipore, Billerica, MA, USA) diluted in PBS with 5 % nonfat milk and 0.1 % Tween-20 (PBST) for 2 hours at room temperature. The membranes were visualized using enhanced chemiluminescence (ECL Western blotting detection reagents; Amersham Pharmacia Biotech, Piscataway, NJ, USA). Actin was used as an internal control.. 2.5 Immunofluorescence staining HMSCs were plated for 24 hours in 48-well culture plates at 2.5 × 103 cells/wells before the experiment. After incubation with or without ION for 24 hours, the hMSCs were cultured in CM, and NCs were cultured in NIM for 21 days. The cells were then washed three times with PBS before fixing in 4 % paraformaldehyde solution (Sigma-Aldrich) in PBS at room temperature for 10 minutes. The cells were then washed twice with PBS and permeabilized with 0.1 % Triton X-100 (Sigma-Aldrich) for 5 minutes. Nonspecific binding sites were blocked using a 2 % BSA solution for 30 minutes at room temperature. hMSCs were incubated with neural primary antibodies including GFAP (1:50) for astrocytes, NeuN (1:100), TH (1:500) and Tuj1 (1:200) overnight at 4°C. The cells were washed and then incubated with fluorescent (FITC/Rhodamine) secondary anti-mouse or anti-rabbit IgG antibodies (Millipore) at room temperature for 45 minutes. The platelets were washed again with PBS, follow by staining with DNA binding dye, 4’,6-diamidino-2-phenylindole (DAPI; 5 µg/mL; Molecular Probes) in PBS for 5 minutes at room temperature29. The cells were then washed twice and imaged using an inverted microscope (Eclipse TS100; Nikon, Tokyo, Japan).. - 10 -.

(18) 2.6 Flow cytometry analysis of neural markers HMSCs were plated 24 hours before the experiment in 6-well culture plates at 1 × 105 cells/well. After incubation with or without ION for 24 hours, the hMSCs were trypsinized and washed three times with PBS. The cells were fixed in 4 % paraformaldehyde solution in PBS at room temperature for 10 minutes, washed twice with PBS and permeabilized with methanol at 4°C for 15 minutes. The non-specific binding sites were blocked with 2 % BSA solution at room temperature for 30 minutes. After centrifugation at 1,500 rounds per minute (rpm) for 5 minutes at 4°C, the cells were re-suspended in PBS. After washing, the cells were labelled with one of the following primary antibodies: GFAP (1:50), NeuN (1:100), TH (1:500) and Tuj1 (1:200) in 2 % BSA solution at 4°C overnight. After washing, the cells were further incubated with secondary fluorescein isothiocyanate (FITC)-conjugated anti-mouse or anti-rabbit IgG (Millipore) at room temperature for 45 minutes. The platelets were washed with PBS and then collected for FL1 measurement. For each group of stem cells, 1,500 counts were measured via FACS Calibur flow cytometry (FACS Calibur; BD Biosciences, Franklin Lakes, NJ, USA) and CellQuest Pro software (Becton Dickenson, Mississauga, CA).. 2.7 Electrophysiological recording A multi-electrode recording MED64 system (Alpha Med Scientific, Japan) was used to observe the changes in hMSC-derived nerve action potentials. Each MED probe contained 64 electrodes in an 8 × 8 grid set point (Alpha Med Science, MED-P515A) and was coated with 5 µg/mL poly-lysine/laminin (Sigma-Aldrich, St. Louis, MO, USA) at 37°C in a 5 % CO2, 95 % air atmosphere for 2 hours. After washing with ddH2O, 1×105 - 11 -.

(19) of cells were seeded onto the MED probes and incubated at 37°C in a 5 % CO2, 95 % air atmosphere overnight. CM and NIM were changed every 2 days for 3 weeks. The multi-electrode recordings used a sampling rate of 50 kHz. Total spikes were counted, and the frequency was analysed with a spike sorting analysis system38.. 2.8 Flow cytometry detection of ION particle uptake HMSCs were plated 24 hours before the experiment in 6-well culture plates at 1 × 105 cells/well. After incubation with or without ION for 24 hours, CM and NIM were changed every 2 days for 3 weeks. hMSCs were trypsinized and washed three times with PBS. ION particle uptake by the cells was determined by the number of SSC measurements. For each group of stem cells, 1,500 cell counts were measured via FACS Calibur flow cytometry (FACS Calibur; BD Biosciences, Franklin Lakes, NJ, USA) and CellQuest Pro software (Becton Dickenson, Mississauga, CA).. 2.9 Magnetic resonance imaging (MRI) MRI was performed using a 7 Tesla animal MR system (Bruker Biospec, 70/30, USR). After ION labelling, undifferentiated and differentiation cells in 6-well plates (1 × 105 cells per well) were collected by trypsinization and then washed, centrifuged, and placed in 300-µl Eppendorf tubes (1 × 105 cells per tubes) in a water tank. T2-rapid acquisition with relaxation enhancement (RARE) pulse sequences were used (TR/TE = 3000/12.276 ms, flip angle = 180°, matrix size = 256 × 256). The slice thickness was 1.0 mm with a 1.0-mm gap. The field of view (FOV) was 80 × 80 mm for coronal scanning of the test tubes and 10 minutes and 40 s for sagittal scanning at the NEX of 5.. - 12 -.

(20) All images were then analysed using the Import Bruker NMR Files and ImageJ software (http://rsb.info.nih.gov/ij/plugins/bruker.html).. 2.10 Intracellular iron content determination The total uptake of Fe by cells was analysed using ICP-MS (Agilent 7500ce, Agilent Technologies, Palo Alto, CA, USA). 1 × 105 cells treated with or without 100 µg Fe/mL ION were collected after 24 hours. The cells were trypsinized, washed and centrifuged. Cell pellets were lysed with 1 ml of 3 % HNO3 (65 % HNO3) acid solution. Samples (10 µl) were diluted in 10 ml acid solution and injected to ICP-MS. The Fe concentration was obtained by interpolating to a standard curve obtained from serial dilutions of 0, 10, 20, 50 and 100 ppb Fe42.. 2.11 Viability assay Cell viability was evaluated by 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide; Sigma-Aldrich Co.) dye assay. Following ION labelling, hMSCs, NCs and unlabelled cells were grown in triplicate in 24-well plates overnight (2.5 × 104 cells/well). Afterward, MTT dye was added to the medium. and the cells were incubated for 1 hour. After incubation, the purple formazan dye generated by viable cells was proportional to the number of viable cells, and absorbance at 570 nm was measured using a microplate reader (Infinite F200; TECAN., Austria).. - 13 -.

(21) 2.12 Reactive oxygen species (ROS) measurements The presence of ROS is an important indicator of oxidative stress. The intracellular ROS was evaluated using dichlorofluorescein diacetate (DCFDA) (Molecular Probes, Eugene, OR, USA) as an oxidation fluorescent probe. 1 × 105 hMSCs or NCs were labelled with 100 µg/mL ION for 24 hours. Cells were washed twice with PBS and incubated in 6-well plates at 37°C for 21 days. Each group was then washed three times with PBS and incubated in culture medium with 10 µM DCFDA at 37°C in the dark for 30 minutes. Cells were then collected and re-suspended in cold PBS. The fluorescent mean intensity (FL-1) of positive cells was analysed using a FACS Calibur flow cytometer (Becton-Dickison, PharMingen USA) with an excitation wavelength of 488 nm and an emission wavelength of 515 nm.. 2.13 Mitochondria membrane potential (MMP) measurements Mitochondrial membrane energization was determined by a lipophilic cationic fluorochrome. dye,. 3,3'-dihexyloxacarbocyanine. iodide. [DiOC6. (3)]. method. (Sigma-Aldrich Co). Briefly, hMSCs and NCs were treated with 40 nM DiOC6 (3) in culture medium and then incubated for 30 minutes at 37°C in the dark. Cells were collected and washed in cold PBS. The fluorescent mean intensity was analysed using a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 501 nm43.. - 14 -.

(22) 2.14 Statistical analysis Data were analysed using SPSS statistical software (Version 12.0; IBM Corporation, Armonk, NY, USA). One-way repeated measures ANOVA and Dunnett's t-test were used to compare the means (n=3). A p-value less than 0.05 was considered statistically significant.. - 15 -.

(23) CHAPTER 3. RESULTS. - 16 -.

(24) Results. 3.1 Differentiated human MSCs exhibited neural-like morphology and neuron markers: Directly labelling hMSCs and NCs with ION To investigate the in vitro differentiation of hMSCs into NCs, hMSCs were incubated in neurogenic induction medium (NIM) for NCs differentiation. Compared with undifferentiated MSCs, NCs exhibited dendrite-like features of long spikes extending into other adjacent cells (Figure 1A) and lower cell densities (Figure 1-1, 1A and 1B). There was no morphological difference between ION-labelled and unlabelled cells under light microscopy. Both hMSCs and NCs incubated with ION had blue dots precipitated inside the cytoplasm, whereas unlabelled hMSCs and NCs did not have blue dots (Figure 1B, arrowhead). TEM images also revealed the presence of internalized ION within the organelles of hMSCs and NCs incubated with ION (Figure1-2, 1C, arrowhead). NCs differentiation was further verified by phosphotungstic acid haematoxylin (PTAH) staining. Additionally, co-staining with Prussian blue revealed iron precipitates inside the cytoplasm. Thin and long dendrite-like structures stained in brown were observed in the NCs. By contrast, cells without neural induction exhibited no axon-like structures, and the cytoplasm was not stained. ION-labelled MSCs and NCs exhibited blue precipitate inside cells (Figure1-2 1D, arrowhead). TEM imaging of the ION structure revealed an inner layer iron-oxide core (Fe3O4, dark black colour) and a non-magnetic outside layer coated with carboxydextran (grey colour) (Figure 2).. - 17 -.

(25) The differentiation of NCs from hMSCs were further evidenced by several neural molecular markers at both the mRNA and protein level (Figure 3). RT-PCR results demonstrated the expression of glial fibrillary acidic protein (GFAP), tyrosine hydroxylase (TH) and NEUROD6 genes at 14 and 21 days after NIM incubation. The mRNA expression of GFAP, TH and NEUROD6 were significantly elevated in the NCs differentiation group regardless of ION labelling. However, no differences in GFAP, TH and NEUROD6 mRNA expression were observed in the hMSCs groups (Figure3-1, 3A). Neuron-specific protein markers GFAP, NeuN, TuJ1 and TH were weakly expressed in undifferentiated MSCs. However, the protein levels of these markers were dramatically expressed in NCs (Figure3-1, 3B). With immunofluorescent staining, the expression of GFAP, NeuN and TuJ1 were visualized as strong fluorescent signals inside the cytoplasm after NIM treatment in NCs groups. ION labelling did not alter the expression of the GFAP (stained 4 in green), NeuN (stained in green) and beta-III tubulin (TuJ1, stained in red) (Figure3-2, 3C). Expression levels of GFAP, TuJ1, and TH were also confirmed after neural-like cell differentiation by FACS analysis (Figure3-2, 3D). The NCs had a significantly higher mean expression of NCs / MSCs (GFAP: 958/168, TuJ1: 707/87, and TH: 637/40) compared with hMSCs with or without (w/o) ION treatment.. 3.2 Electrophysiological function The electrophysiological characteristics of ION-labelled MSCs and NCs are shown in Figure 4. The ability acquired by NCs differentiated from MSCs to generate spontaneous firing activity patterns was not altered after labelling with ION (Figure 4A). The quantitative results and spike frequency of the cells with neural-like morphologies indicated the active membrane properties of the cells, as shown in Figure 4B. - 18 -.

(26) There was no significant difference between NCs with or without ION labelling. Moreover, the voltage of each action potential signal was higher in the cells in the NCs group (90 mV) than in cells that did not undergo NCs differentiation (50-60 mV) (Figure 4A, 4B) Labelling with ION did not alter the voltage amplitude of the spontaneous firing activity. By contrast, MSCs did not express significant spontaneous firing activity patterns. In the 180-s observation period, the cells that differentiated into neural-like cells generated more spikes (80 times/180 s) than the cells that did not undergo differentiation (10-30 times/180 s). Taken together, these data demonstrated that NCs differentiated from MSCs have key features of functional neurons with the long-term ability to generate spontaneous firing activity patterns.. 3.3 In vitro determination of ION uptake by MRI, inductively coupled plasma mass spectrometry (ICP-MS) and flow cytometry We next examined whether the ION-labelled cells can be detected under non-invasive MRI. Under T2-weighted images, ION-labelled NCs and MSCs showed dark dots at the bottom of the test tube, whereas no dark signal was detected in cells without ION labelling (Figure 5A). The iron content of the cells was determined using ICP-MS (Figure 5B). The intracellular level of iron was significantly greater in MSCs and NCs treated with ION (29.2±1.5 pg/cell and 25.9±2.0 pg/cell, respectively) than in untreated MSCs and NCs (0.38 pg/cell and 0.93 pg/cell, respectively). Cells granularity determined by flow cytometry showed more side scatter counts (SSCs) in the ION-treated cells than in the untreated cells (Figure 5C).. - 19 -.

(27) 3.4 Cell behaviour Cytotoxicity. testing. of. ION-treated. cells. was. verified. by. using. 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT), mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) production assays (Figure 6). Cells viability assay showed a significant increase in formazan formation in ION-labelled NCs but not in ION-labelled MSCs (Figure 6A). There was no significant alteration in the MMP of hMSCs and NCs labelled with IONs. ION-labelled hMSCs and NCs displayed elevated ROS production compared to unlabelled hMSCs and NCs (Figure 6B).. - 20 -.

(28) CHAPTER 4. DISCUSSION. - 21 -.

(29) 4. Discussion The loss of neurons after brain injury is irreversible and different methods to preserve brain functions were developed. Tissue engineering offer one way to reconstruct the lost neuron and limite success was witnessed. We developed differentiating methods from mesenchymal stem cells into neurons which is capable of been visualized under MRI, which is critical in the neuron transplantation.. 4.1 Cell behavior The labeling of the IONs toward different kinds of cells such as MSCs, macrophage has been investigated38,44,45. Although there is some debate on the influence of chondrogenic differentiation capacity after another Feridex, another IONs that used in USA, there is no differentiation capacity change after IONs in our studies38,44. The observation of differentiated cells in vivo can last up to three weeks, which is sufficient for the observation of cell implantation and migration. Moreover, there is no need to sacrifice these animals in each observataion, and thus increase the efficacy and power of each study. In this study, we demonstrated that ION labelling does not affect the differentiation capability of hMSCs to NCs. Cellular functions, including morphology, viability, oxidative stress, and mitochondrial member energization, of ION-labelled hMSCs and NCs were intact. IONs induced no significant difference in mRNA, protein content, neuron-specific protein marker expression, spontaneous firing activity patterns, or - 22 -.

(30) intracellular iron levels in NCs. The clinically used ION, ferucarbotran, has a high uptake capability by stem cells and can be detected using the 1.5 T MRI at the single-cell level38,42-44,46,47. It has been known that IONs can be uptaken into neuron progenitor cell and been visualized in MRI up to 7 days29. Recent study also demonstrated that Resovist has higher uptake capability17,40,42,43,48,49. We chose Resovist not only because it has higher uptake capacity for cells, it is also a clinical available medicine which might relieve the worry of its cytotoxicity. Our study revealed a slight increase in cell growth after ION labelling possibly because the cell cycle is involved after intracellular Fe release from lysosomes18. The ROS of labelled cells were slightly increased due to the increase in H2O2 in the cytoplasm10,44,50,51.. These results are consistent with the findings of Chen et al29. Similar to the previous findings19,20, we found no influence of ION uptake on the MMP. We noticed neuron differentiation protein expression such as neuron specific protein from 2nd to 3rd weeks.. 4.2 Different sizes of supraparamagnetic ION Different sizes of supraparamagnetic ION have been used in labelling and tracking neural-like cells during differentiation using MR imaging methods. Such ION include ultrasmall superparamagnetic iron oxide (USPIO) particles (≤30 nm in diameter), superparamagnetic iron oxide (SPIO) particles (30–200 nm in diameter), and micron-sized superparamagnetic iron oxide (MPIO) particles52. After intravascular injection, USPIO enters the 6 capillaries, then the matrix, and ultimately arrives at the lymph nodes, but most SPIO accumulates in the reticular endothelial system53. Crabbe et al compared the labelling efficiency of three different stem cell populations mouse. - 23 -.

(31) embryonic stem cells (mESCs), rat multipotent adult progenitor cells (rMAPCs), and mouse. mesenchymal. stem. cells. (mMSCs). with. three. different. (ultra)small. superparamagnetic iron oxide [(U)SPIO] particles (Resovist, Endorem, Sinerem). The labelling efficiency with Resovist and Endorem significantly differed among stem cells. They found that the minimum cell density that needed to be detected by the MR imager was 75 cells/µl in Sinerem ® (USPIO, 20 nm), and 5 cells/µl in Endorem ® (SPIO, 80-150 nm) and Resovist ® (SPIO, 60 nm). After labelling with Resovist, rMAPCs can still express the neuroprogenitor genes Sox2 and Pax6. They also observed migration to the injured area 3 weeks after implanting 10,000 Resovist-labelled rMAPCs in the bilateral striatum48. Based on this previous research, we sought to label MSCs and NCs with SPIO, i.e., Resovist. Many studies have shown no morphological changes in MSCs, neural stem cells (NSCs), or neural progenitor cells (NPCs) labelled with MPIO, and all cell types expressed TH, TuJ1, and nestin after MPIO labelling. As MPIO particles are larger, they are more easily detected, especially at the single-cell level. Another advantage of MPIO particles for cellular tracking is that they have a longer half-life than (U)SPIO particles in the body. Evaluation of intracellular labeling with micron-sized particles of iron oxide (MPIOs) as a general tool for in vitro and in vivo tracking of human stem and progenitor cells54. However, (U)SPIO particles can be metabolized over the long-term, whereas MPIO particles with a size >100 nm cannot. Ferucarbotran is an ION with a particle size of approximately 60 nm and is composed of an iron oxide core coated with carboxydextran to prevent nanoparticle aggregation. The iron oxide is metabolized and reused as a haemoglobin component. Carboxydextran is degraded by lysosomes38. A recent study demonstrated that, after exposure of human neuroblastoma cells to 10 µg/ml of 10 nm ION for 24 h, obvious neurotoxicity, including decreased cellular dopamine, increased ROS, increased neural α-synuclein and activated tyrosine kinase c-Ab1 expression, and inhibition of cell-proliferation, were observed in - 24 -.

(32) neural cells55,56 . Another study investigating the size and relationship of ION retention in the central nervous system concluded that 40-nm ION possess more evident detention properties in the CNS than 280-nm ION56. Another type of ION, the Molday ION, have also been introduced, which are new ultra-small superparamagnetic ION with a magnetic core and hydrodynamic sizes of approximately 8 and 35 nm, respectively, conjugated with Rhodamine-B (Rh-B) (2 fluorophores 7 per particle). The Molday Rhodamine-B ION significantly reduced the survival, proliferation, and differentiation rate of neural stem cells, and upregulated the immune response in recipient animals in a concentration of 50 µg/mL30. Such characteristics are not suitable for use as a labelling agent. We speculate that this might be the result of the very small sizes of the particles, the coating material and its fluorophore ligands. These support the possibility that ferucarbotran may be one of the best ION for labelling cells for studies of the nervous system. We selected Resovist ION because Resovist is a clinically used MR contrast medium approved by the FDA. Internalization can be performed without any transfection agent. Resovist has several advantages such as its good biocompatibility, low toxicity, and excellent stability31 . Using Resovist labelling, studies of rabbit MSCs revealed that it induced no changes in morphology, it had no effect on neural differentiation, and the protein expression levels of the mature neuron markers TuJ1 and NSE were maintained32,33. These findings are similar to our results reported here. Another report using human amniotic membrane-derived MSCs (hAM-dMSCs) showed no expression of GFAP protein, although there was no resulting morphological change. This finding might be due to the fact that glial formation cannot be detected in a relatively short period of incubation during neural-induction differentiation (7 days) or that a different source of MSCs was used31. - 25 -.

(33) 4.3 NPs coating To improve biocompatibility and biodistribution and to prevent precipitation and agglomeration under physiological conditions, SPION particles are coated with an amphiphilic layer of polymers. These polymers can be hydrophilic and protect the iron oxide core from degradation34. Resovist is coated with carboxydextran, which is a type of natural polysaccharide with a net charge, water solubility, biocompatibility, and biodegradability and is enriched with hydroxyl groups, which have been reported to interact with iron oxide via hydrogen bonding. In addition, poly(L-lysine) (PLL), a positively charged peptide, can be used to facilitate cellular internalization. Ferumoxides-PLL complex-labelled MSCs does not alter biochemical or haematological measures of organ function35. Albukhaty et al reported that a mixture of 25 µg/0.75µg ml SPIO/PLL enhanced cellular activity after differentiation36. Ke et al labelled bone marrow stroma cell-derived neural stem cells (BMSC-D-NSCs) with PLL-coated Feridex and found no morphological changes after differentiation; NSC and GFAP expression were also preserved. After 8 autologous implantation into the monkey brain, BMSC-D-NSCs were still locally detected by MRI as black dots, and they could express NSE protein according to immunohistochemistry assays37. Delcroix. and. associates used. another negatively charged. material,. 1-hydroxyethylidene-1.1-bisphosphonic acid (HEDP)-coated SPIO particles, to label rat MSCs and found no morphological changes after differentiation and preserved TuJ1 and NeuN expression39.. - 26 -.

(34) Chen et a47 labelled mouse NSCs with hydrogen-terminated ultra-nanocrystalline diamond (H-UNCD) and found that it causes spontaneous neural differentiation of NSCs. They claimed that the characteristics of wear/corrosive resistance in this crystalline structure might improve cellular adhesion and extension. This was proven in their scanning electronic micrograph study, which showed that the filopodia of NSCs extended more than 100 nm and that the expression of neuron markers such as GFAP and TuJ1 was increased according to immunofluorescence staining47. As the underlying mechanism, they proposed that the crystalline structure can increase the linkage between NSCs and cytokine or integrin release, which is not related to the hydrophobicity. The above studies provide insight into some particular surface structures, such as UNCD, that might have an impact on the differentiation of NSCs, but the charge does not seem to be an important factor. After labelling hMSCs with ferumoxides, another type of ION, the iron content inside the hMSCs decreased upon cell division57. Both decreased iron content and loss of cellular granularity are correlated with a decrease in the long-term detectability of cells by MRI58. In a clinical MRI study of traumatic brain injury patients in which autologous neural stem cells were labelled with ferumoxides and transplanted into the brain injury area, strong signal changes were detected from days 1 to days 1459. After 2 weeks, the cells began to move around the damaged area, and the signal weakened. No signal was detected until the 7th week, which may be related to the fact that the calibrated neural stem cells had moved and were scattered in relation to the localization region. Consequently, the detectability of ION-labelled neurons is dependent on the initial iron oxide labelling concentration and the speed of cell division. Higher initial iron concentration might influence cell behaviour that is averse to neuron repair. Our study shows that up to 100 µg Fe/mL for incubation is still within the safe range and has little - 27 -.

(35) influence on cell viability and differentiation. The intracellular levels of iron in hMSCs and NCs treated with ION were 29.2±1.5 pg/cell and 25.9±2.0 pg/cell, respectively, in the current study, which are comparable to our previous report (23.4 pg/cell in ION of 100 µg Fe/mL)38 and slightly higher than the levels reported in another study (15–20 pg Fe/cell in ION of 20 and 30 µg Fe/ml)48. This suggests that MR imaging using our ION-labelling protocol can successfully localize the stem cells and can be used to track their persistence and migration over time in animal models. Although a previous report claimed nearly 100 % labeling efficiency of ION by Prussian Blue iron stain alone, no quantitative evidence was provided to support the conclusion60. The implantation of MSCs into rats that underwent spinal cord injury or stroke improved their performance in previous studies. However, MSCs implanted into the rat brain do not differentiate into functional neurons 61-64. Similarly, the implantation of adult NCs failed to improve cell function and migration, which are critical in neuron repair65. However, we showed that the induction of MSCs by growth factors is morphologically effective. Moreover, the migration of cells can also be monitored by non-invasive MRI methods. The combination of growth factor induction and ION labeling will facilitate future research in neuron tissue engineering.. - 28 -.

(36) 4.4 Iron Content Pawelczyk et al. 57,66. found decreased iron content inside the BMSCs after labeling. BMSCs with ferumoxides after cell devision. We analysed the iron content by ICP-MS and the granularity of each cytoplasm also has similar effect. Both decreased iron conent and loss of cellular granularity correlate with the detectability of cells by MRI44,67 . Conseqeuntly, the detectability of IONs labeled neurons are depedent on the initial iron oxide labeling concentration and the speed of cell devision. Undoubtly, higher initial iron concentration might influence the cell behavior that is adverse to the neuron repair. Our study shows up to 100 µg Fe/mL of incubation concentration is still within the safe range that has little influence on cell devision and differentiation.. 4.5 Neural induction medium (NIM) There are many ways to differentiate cells into neurons. Growth factor or other chemicals68,69 has been tried and yields good result. Growth factos such as basic fibroblast growth factor (b-FGF), epidermal growth factor (EGF) platelet-derived growth factor (PDGF) are important for cells differentiating into neurons70-72 and is included in our differentiation medium. We also use brain-derived growth factor (BDGF)73-77 and retinoic acid (RA)78-80 to promote dendrite growth and transform its spindle shape into neuron like cells.. - 29 -.

(37) 4.6 NCs NCs derived from rabbit BM-MSCs did not affect the morphology of neurons and expressed specific neuroprotective proteins (NSE, MAP) after labelling with ferucarbotran33. Whole-cell patch-clamp recordings showed that these NCs exhibited electrophysiological activity. Our labelled hMSCs differentiated into NCs that exhibited significant levels of mature neural biomarkers, including observable dendrites and spontaneous firing activity patterns. Neural function still cannot be restored in patients suffering from dementia, Parkinson’s disease, and stroke even despite aggressive treatment methods. The treatment of these disorders with conventional methods or medication can partially relieve symptoms. Furthermore, the spontaneous generation of neural cells after brain injury is limited. Cell therapy can provide a chance to regain neuronal function by replacing dead or degenerative neurons with newly differentiated cells9,81-88. There is increasing evidence that inducted neuron like cells which may be implanted into the spinal cord of rats suffered from spinal cord injury neuron markers. 89-94. .The differentiated cells exhibit higher level of. 95-98. . Another group showed improved auditory function after cochlear. damage by implanting neuron stem cells cultured and inducted in vitro. 99,100. . These data. indicate the in vitro stem cell induction methods may has the capability of replace damaged neuron tissue, causing improvement of the function status.. - 30 -.

(38) The induction of pluripotent stem cells that originate from human fibroblast has drawn much attention in cell replacement therapy due to the autologous origin of the fibroblasts. However, the tumorigenicity found in phase I clinical trials has limited the applications of this technique101. However, hMSCs have extensive differentiation capacity without inducing tumorigenicity, they are easy to propagate, and they exhibit immunomodulatory properties that are beneficial in damaged brain tissue102. However, whether these differentiated NCs retain their immunomodulatory capacity should be further evaluated. Induced neural-like cells implanted into the spinal cords of rats that underwent spinal cord injury improved neurogenesis in rat models and improved function in models of Parkinsonism84,86. Such induced neural cells exhibited 10 higher levels of neural markers88,103. It is important to be able to confirm whether hMSCs can express the proteins of nerve cells before or during implantation in vivo and in pre-clinical trials. In our study, the ION-labelled NCs expressed multiple mature neural protein markers and produced many NCs in vitro, and thus, they could possibly be used to replace irreparably damaged nerve cells.. 4.7 Neural Protein expression Neuron-specific nuclear (NeuN) protein is a neucleoprotein which expresses in neurons and this protein could be found in neuron induction which is conducted by Sanchez-Ramos. 73. . Our study also shows NeuN expression in the 3rd week of neuron. induction. The addition of Resovist didn’t alter the expression status of NeuN.. - 31 -.

(39) Another indicator, Glial fibirllary acidic protein (GFAP),. exsits in astrocytes as. structural protein which could be found mostly 14 days after induction57,104. Our study also demonstrated GFAP expression in cells with neuron induction no matter Resovist were added or not. Beta-III tubulin is responsible for neuron microtubule formation. Tyrosine hydroxylase are related to dopaminergic neuron. In our induction study, these two proteins are visualized no matter Resovist incubation or not. NSE could only be found in mature neurons and our study showed positive expression in neuron induction groups.. 4.8 Spontaneous firing frequency Although there is evidence that implantation of MSCs into rats suffered from spinal cord injury or stroke may increase the performance status of affected rats87,10584,85,106, some researcher found the MSCs implanted into rat’s bain will not differentiate into functional neurons40,87,88. However, implantation of adult NCs failed to prove cell function and migration ability which are critical in the neuron repair98,107. To facilitate the cell migration, differentiation, induction of MSCs by chemical or growth factor methods were used. The determining factors are cell surface markers, microscopic morphological appearance such as dendrite and axons38,108,109. Our study shows the induction of MSCs by growth factors are morphologically effective. The biomarkers of neurons are expressed well and more importantly, increased firing amplitude and firing frequency110,111 may fulfill the needs for neuron repair. Moreover, the migration of cells can also be monitored by non-invasive MR methods. The combination of growth factor - 32 -.

(40) induction and labeling with IONs should facilitate the future research in the neuron tissue engineering.. - 33 -.

(41) CHAPTER 5. CONCLUSION. - 34 -.

(42) 5. Conclusion We established an ION-labelling and MRI-based protocol for studying neural stem-like cells differentiation and hMSC-derived NCs. All the derived NCs exhibited significant mature neural biomarkers, including observable dendrites and spontaneous firing activity. In ION labelling, the process of implantation and cell migration can be traced by MRI, which is ideal for the analysis of implanted cells in the damaged regions and can shed light on cell therapies applied to the central nervous system.. - 35 -.

(43) CHAPTER 6. REFERENCES. - 36 -.

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(64) CHAPTER 7. FIGURES. - 57 -.

(65) Figure 1-1 Comparison of hMSCs differentiation capacity into NCs with or without (w/o) ION. Light microscopic image (A), Prussian blue staining (B). The blue and black dots indicated by black arrows are ingested ION.. - 58 -.

(66) Figure 1-2 Comparison of hMSCs differentiation capacity into NCs with or without (w/o) ION. TEM image (C) and co-staining with PTAH and Prussian blue (D). The blue and black dots indicated by black arrows are ingested ION.. - 59 -.

(67) Figure 2 TEM images of ION (Resovist, ferucarbotran) Different concentrations of Resovist solution (i) colour images; left: 10 µg/ml, right: 100 µg/ml) magnetized using a permanent magnet. Particle size was 45-60 nm (ii).. - 60 -.

(68) Figure 3-1 Characterization of neural differentiation markers in hMSCs treated with or without neural induction medium after ION labelling. (A) Comparison of neural marker expression (GFAP, TH, and NEUROD6) by RT-PCR after induction of neural-like cell differentiation with or without ION labelling. (B) Western blot analysis showing that hMSCs incubated with NIM could be differentiation into NCs and increase the protein expression of TH, NeuN, GFAP, TuJ1 and NSE. Actin as an internal control.. - 61 -.

(69) MSCs W/O. NCs. IONs. W/O. IONs. C NeuN. GFAP TuJ1. D. FL-1. Figure 3-2 Characterization of neural differentiation markers in hMSCs treated with or without neural induction medium after ION labelling. (C) Immunofluorescent staining of h-NeuN and GFAP in green, TuJ1 in red, and nuclei in blue (DAPI). (D) Flow cytometry analysis of GFAP, TH and TuJ1 of the hMSC-derived neural cells with or without ION labeling.. - 62 -.

(70) - 63 -.

(71) Figure 4 Action potentials of hMSCs, NCs with or without ION labelling. (A) Quantitative analysis of the action potentials (mV, millivolt; ms, milliseconds). (B) The number of spikes are reported. (C) Both action potential amplitude and the number of spikes increased after neural-like cell induction. ION labelling had no influence on the action potential amplitude or number of spikes. Quantitative analysis of the action potentials were represented as mean ± S.E. of three independent experiments. Comparisons between the groups were performed using repeated measures analysis of variance (One-way ANOVA). Whereas differences between means were inspected with Dunnett’s multiple comparison post-tests. * p <0.05.. - 64 -.

(72) Figure 5-1 Quantification of iron content after labelling with or without ION before and after induction of neural-like cell differentiation. (A) In vitro MRI, (B) ICP-MS. In vitro MRI also demonstrated that the labelling efficiency could be visualized by clinical MRI. MR image were represented as mean ± S.E. of three independent experiments. Comparisons between the groups were performed using repeated measures analysis of variance (One-way ANOVA). Whereas differences between means were inspected with Dunnett’s multiple comparison post-tests. * p <0.05.. - 65 -.

(73) MSCs. SSC. Figure 5-2 Quantification of iron content after labelling with or without ION before and after induction of neural-like cell differentiation. (C) SSCs based on flow cytometry of each group. We observed increased iron content in ferucarbotran-labelled cells and slightly increased SSCs in flow cytometry, indicating increased granularity in ION-labelled cells. Comparisons between the groups were performed using repeated measures analysis of variance (One-way ANOVA). Whereas differences between means were inspected with Dunnett’s multiple comparison post-tests.. - 66 -.

(74) Figure 6-1 Measuring cell behaviour using three different assays. (A) Cell viability (MTT) assay Human mesenchymal stem cells with or without 21 days of differentiation induction were treated with or without 100 µg/mL ION for 24 hours. Cell viability assays were represented as mean ± S.E. (n=3). Comparisons between the groups were performed using repeated measures analysis of variance (One-way ANOVA). Whereas differences between means were inspected with Dunnett’s multiple comparison post-tests. * p <0.05.. - 67 -.

(75) Figure 6 Measuring cell behaviour using three different assays. (B) mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) assay (FL-1, fluorescent mean intensity). Human mesenchymal stem cells with or without 21 days of differentiation induction were treated with or without 100 µg/mL ION for 24 hours. The data is represented as means ± S.E.M of three independent experiments. Comparisons between the groups were performed using repeated measures analysis of variance (One-way ANOVA). Whereas differences between means were inspected with Dunnett’s multiple comparison post-tests.. - 68 -.