Evaluation effects of lingzhi mushroom (ganoderma lucidum) on neural stem cells isolated from embryonic mouse brain (mus musculus var. albino)

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MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY EVALUATION EFFECTS OF LINGZHI MUSHROOM (Ganoderma lucidum) ON NEURAL STEM CELLS ISOLATED FROM EMBRYONIC MOUSE BRAIN (Mus musculus var. albino) SUPERVISORS STUDENT Dr. PHAM VAN PHUC NGUYEN THI MAI DAN MSc. TRUONG HAI NHUNG Student ID code: 3082584 Session: 34 (2008- 2013) Ho Chi Minh City, 05/2013 APPROVAL SUPERVISORS Dr. PHAM VAN PHUC STUDENT NGUYEN THI MAI DAN MSc. TRUONG HAI NHUNG Can Tho, PRESIDENT OF EXAMINATION COMMITTEE ABSTRACT Neurodegenerative diseases or neurological damages such as Parkinson, Alzheimer, Huntington and spinal cord injury have become a great challenge to public health. In fact, treatment for these diseases is restricted since new neural cells cannot be generated by the nervous system, thus an efficient therapy is emergently required. Recently, stem cells, especially neural stem cells (NSCs), are employed as an innovative tool for the replacement of damaged cells due to self-renewal as well as pluripotent capacity. NSCs were isolated from 13.5 – 15.5 post coitum embryonic mouse brain and cultured in DMEM/F12 serum-free medium modified with B27, N2, heparin, EGF and FGF at 37o C, 5% CO2. Candidate cells were identified for neural stem cell characteristics by neurosphere assay, the expression of Nestin, CD133, Sox-1 and GFAP. Those NSCs were then cultured in 96well plate added with Ganoderma lucidum extract at 100µg/ml, 500µg/ml, 1000µg/ml for the evaluation of medicinal effects of such mushroom on cell proliferation. The results revealed that 72% of sample was successfully cultured, cells isolated from 13.5-15.5 day post coitum fetus showed the best capacity of culture, isolated cells could formed neurosphere, expressed Sox1, CD133, Nestin and GFAP. Extract at 500µg/ml showed the best effects on neural stem cell growth. Keywords: Ganoderma lucidum, neurosphere assay, neural stem cell markers, neurosphere size. i CONTENTS ABSTRACT ...............................................................................i CONTENTS ..............................................................................ii CHAPTER 1: INTRODUCTION ............................................. 1 CHAPTER 2: MATERIALS AND METHODS ..................... 3 2.1 Materials ............................................................................... 3 2.2 Methods ................................................................................ 5 2.2.1 Activity 1: Isolation and culture of candidate NSCs isolated from embryonic mouse brain (Mus musculus var. albino)...................................................... 5 2.2.1.1 Isolation and primary culture of cells isolated from embryonic mouse brain .................................... 5 2.2.1.2 Secondary culture of candidate NSCs ........................ 5 2.2.2 Activity 2: Determination of characteristics of isolated candidate cells.................................................. 6 2.2.2.1 Neurosphere assay .................................................... 6 2.2.2.2 Expression of markers of candidate NSCs ................. 6 2.2.2.2.1 Determination of Sox1 expression of candidate NSCs by RT-PCR (reverse transcription polymerase chain reaction) ......... 6 2.2.2.2.2 Determination of CD133 expression of candidate NSCs by flow cytometry ................. 7 2.2.2.2.3 Determination of Nestin expression of candidate NSCs by immuno - cytochemistry ................................................. 7 2.2.2.3 Differentiation to astrocytes and GFAP expression.... 8 ii 2.2.3 Activity 3: Evaluation effects of Ganoderma lucidum on cell proliferation ......................................... 9 2.2.4 Data analysis ................................................................... 9 CHAPTER 3: RESULTS AND DISCUSSION ...................... 10 3.1 Isolation and culture of candidate NSCs from embryonic mouse brain ..................................................... 10 3.1.1 Isolation and primary culture of candidate NSCs............ 10 3.1.2 Secondary culture .......................................................... 11 3.2 Determination of characteristics of candidate NSCs ............. 12 3.2.1 Neurosphere assay ......................................................... 12 3.2.2 Expression of markers of candidate NSCs...................... 13 3.2.2.1 Sox1 expression of candidate NSCs......................... 13 3.2.2.2 CD133 expression of candidate NSCs ..................... 14 3.2.2.3 Nestin expression of candidate NSCs ...................... 15 3.2.2.4 Differentiation and GFAP expression of candidate NSCs ...................................................... 16 3.3. Effects of Ganoderma lucidum on neural stem cell proliferation ...................................................................... 17 CHAPTER 4: CONCLUSION AND SUGGESTION ............ 20 4.1 Conclusion .......................................................................... 20 4.1.1 Isolation and culture of NSCs ........................................ 20 4.1.2 Neural stem cell characteristics determination ................ 20 4.1.3 Cell proliferation effects of Ganoderma lucidum extract .......................................................................... 20 4.2 Suggestion .......................................................................... 20 REFERENCES ....................................................................... 22 iii CHAPTER 1: INTRODUCTION Neurodegenerative diseases, the quantity loss of functional neural cells in the brain, spinal cord and nerves due to apoptosis or necrosis, could affect body movement, understanding, respiration, emotion and memory. According to WHO (World Health Organization), there were 1 billion patients suffering neurodegenerative diseases in 2007, with 50 million cases of epilepsy and 24 million cases of Alzheimer, resulting in 6.8 million cases of death annually. Thus, neurodegenerative diseases, with their dramatic growth, have been raising a great concern among general public over the past 65 years. Among these, Alzheimer’s disease and Parkinson’s disease are the most popular neurological disorders in the world. Such diseases have been treated by medication or conventional surgery; however, many side-effects of medicine were addressed. In the same context, surgery was reported as an inefficient therapy since patients had to undergo surgery for several times, but some symptoms still remained. Recent studies have discovered potential uses of stem cells in treating such disorders, especially Parkinson’s disease due to its enormous efficacy. However, stem cell population is limited in mature tissues, thus embryonic stem cells with higher selfrenewal and pluripotent capacity are employed. Besides, neural stem cells (NSCs) from endogenous sources do not efficiently migrate to specific regions in brain for replacement demand. As a consequence, extrinsic factors are used to trigger appropriate signals for cell proliferation and differentiation (Reimers, 2008). 1 The study “Evaluation effects of Ganoderma lucidum on neural stem cells isolated from embryonic mouse brain (Mus musculus var. albino)” was conducted to clarify medicinal effects of such mushroom on cell proliferation at in vitro level and set up a foundation for further research. Objective: - Isolating and culturing neural stem cells from embryonic mouse brain. - Determining the best concentration of Ganoderma lucidum for neural stem cell growth in vitro. 2 CHAPTER 2: MATERIALS AND METHODS 2.1 Materials Period: December, 2012 – May, 2013 Location: Stem Cell Research and Application Laboratory, Ho Chi Minh University of Science, Vietnam National University. Sample: cells from embryonic mouse brain (Mus musculus var. albino). 13.5-15.5 day post coitum mouse were purchased from Pasteur Institute, Ho Chi Minh City. Ganoderma lucidum extract was purchased from National Institute of Medicinal Materials, Ho Chi Minh City. Chemicals: - Phosphate buffered saline (-) - PBS modified with Streptomycin and Penicillin (1X, 5X, 10X) - Trypsin/EDTA solution - RT-PCR chemicals: Primers (Sox1, GAPDH), One-step RTPCR pre-mix (iNtRON Biotechnology) - Electrophoresis chemicals: 1.5% agarose gel, 0.5X TAE buffer - Immunocytochemistry chemicals: + Anti-Nestin antibody conjugated with FITC + Anti-CD133 antibody conjugated with FITC + Anti-GFAP antibody conjugated with Rhodamine + Hoechst 33342 - Flow cytometry chemical: FACS Fluid 3 - NSC medium Chemicals Concentration H-DMEM/F12 94.3ml/l 30% Glucose 2 ml/l Hepes buffer 1M 0.5 ml/l Progesterone 1000X 0.1 ml/l Putrescine 1000X 1 ml/l EGF 20 ml/l FGF 20 ml/l Antibiotics 1 ml/l N2, B27 growth supplement (Invitrogen, USA) - Differentiation medium Chemicals Concentration H-DMEM/F12 89ml/l Heparin 25µg/ml Insulin 5µg/ml Transferrin 5µg/ml Gentamycine 1ml/l N2, B27 growth supplement (Invitrogen, USA) 4 2.2 Methods 2.2.1 Activity 1: Isolation and culture of candidate NSCs isolated from embryonic mouse brain (Mus musculus var. albino) 2.2.1.1 Isolation and primary culture of cells isolated from embryonic mouse brain NSCs were collected from 13.5-15.5 day post coitum fetus by abdominal surgery and kept in 10X PBS solution. Mouse fetus was decontaminated in 10X, 5X and 1X PBS solution, respectively. Those fetuses were then decapitated and washed by 1X PBS before brain isolation. Isolated mouse brain was mechanically dissected, suspended and filtered through a 70µm cell strainer. The dissociated cells were collected by centrifugation at 2500rpm for 5 minutes and were resuspended in 1ml free-serum NSC medium. Such cells were cultured in 6-well plates and incubated at 37oC and 5% CO2. 2.2.1.2 Secondary culture of candidate NSCs Since living cells consumed nutrients, increased in size and produced waste, which could be toxic to themselves, during proliferation; thus, cell passages were taken to remove toxic substances, provide nutritious elements and space for cell growth. After 2-3 days of culture, suspended neurospheres were collected by centrifugation at 1500rpm for 5 minutes, mechanically dissociated and resuspended in 2ml NSC medium, followed by being subcultured in new wells at 37oC, 5% CO2. 5 2.2.2 Activity 2: Determination of characteristics of candidate NSCs 2.2.2.1 Neurosphere assay Neurospheres of passage 4 were collected by centrifugation at 1500rpm for 5 minutes, resuspended in 2ml NSC medium and mechanically dissociated. These single cells were then counted by NucleoCounter and plated at a density of 104 cells/ml in 96-well plate at 37oC, 5% CO2. Cell proliferation was observed and evaluated under inverted optical microscope every 24 hours. 2.2.2.2 Expression of markers of candidate NSCs 2.2.2.2.1 Determination of Sox1 expression of candidate NSCs by RT-PCR (reverse transcription polymerase chain reaction) For RT-PCR analysis, spheres of passage 4 were collected by centrifugation at 1500rpm for 5 minutes prior to RNA extraction. RNA was then extracted by using easy-BLUE kit (iNtRON Biotechnology) and RT-PCR was performed with Onestep RT-PCR premix kit (iNtRON Biotechnology). Expression level of Sox1 was measured by electrophoresis on 1.5% agarose gel (110V, 30 minutes). Table 3.1 RT-PCR mix No. Components Concentration Volume 1 RNA 500ng/µl 1.0µl 2 H2O - 5.5µl 3 One-step RT-PCR pre-mix - 7.5µl 4 Primers (forward and reverse) 0.25mM 1.0µl 6 70oC 42oC 60 mins 5 mins 95oC 95oC 10 mins 15 mins toC gradient 30 secs 72o C 30 secs 72o C 10 mins 4oC ∞ 30 cylces Figure 3.4 Thermal cycle of RT-PCR 2.2.2.2.2 Determination of CD133 expression of candidate NSCs by flow cytometry Spheres were collected by centrifugation at 1500rpm for 5 minutes and mechanically dissociated to obtain a single-cell suspension in 1ml 0.25% Trypsin/EDTA solution. Cells were incubated at 37oC for 2 minutes and harvested by centrifugation (2500rpm, 5 minutes). Pellet was rinsed in PBS solution, then 100µl FACS Fluid and 5µl CD133 antibody were added with the ratio 1:200, respectively. This solution was incubated on an orbital shaker at room temperature for 15 minutes or at 4 oC for 30 minutes. Centrifugation (2500rpm, 5 minutes) was performed to harvest single cells, then cells were resuspended in 500µl FACS Fluid solution. Cell sorting was performed on FACS Calibur flow cytometer (BD Science). 2.2.2.2.3 Determination of Nestin expression of candidate NSCs by immunocytochemistry Spheres were preincubated in 1ml 1% BSA solution at room temperature for 10 minutes, followed by washing with PBS and centrifugation (1500rpm, 5 minutes). These neurospheres 7 were fixed by FCM fixation buffer and incubated on a rotator at room temperature for 30 minutes, then they were washed and centrifuged (1500rpm, 5 minutes) before being permeabilized in 1ml FCM permeabilization buffer for 5 minutes. Buffer was removed by centrifugation (1500rpm, 5 minutes) and spheres were resuspended in a solution containing anti-Nestin antibody labeled with FITC and Hoechst 3342 at room temperature for 60 minutes or at 4oC overnight in dark conditions. Spheres were then harvested by centrifugation, resuspended in 1ml PBS and plated in 24-well plate for fluorescently microscopic observation. 2.2.2.3 Differentiation to astrocytes and GFAP expression * Differentiation: Spheres were harvested by centrifugation at 1500rpm for 5 minutes and resuspended in 2ml differentiation medium. Suspension of cells was cultured at 37oC, 5% CO2 for 10 days. * Immunocytochemistry: Differentiation medium was removed and cells were fixed by 100µl FCM fixation buffer, followed by incubation on a rotator at room temperature for 30 minutes. After fixation, cells were stained by anti-GFAP antibody labeled with Rhodamine and Hoechst 33342 at room temperature for 60 minutes or at 4oC overnight in dark conditions. Fluorescent microscopy was employed to observe stained cells. 8 2.2.3 Activity 3: Evaluation effects of Ganoderma lucidum on cell proliferation Neurospheres, of which diameters were 100-250µm long, were plated in 96-well plate (1-2 spheres/well) and cultured in NSC medium modified with G.lucidum extract at different concentration: 0µg/ml (Control), 100µg/ml, 500µg/ml and 1000µg/ml (37oC, 5% CO2). Each treatment was repeated 3 times. The experiment with 1 factor was arranged randomly. Criteria of evaluation: changes in neurosphere sizes were recorded every 24 hours for 4 days by observing under inverted optical microscope. 2.2.4 Data analysis Changes in neurosphere sizes were reported as mean ± standard deviation (SD) with 95% of confidence. Differences among means were analyzed by T-test, using Microsoft Excel 2007 software. 9 CHAPTER 3: RESULTS AND DISCUSSION 3.1 Isolation and culture of candidate NSCs from embryonic mouse brain 3.1.1 Isolation and primary culture of candidate NSCs Among 14 samples, 10 samples were successfully cultured with the proportion of 72%. NSCs isolated from the 13.5-15.5 day post coitum embryonic brains, especially E14 cells, show the best rate of proliferation and sphere formation since during this period, receptors for growth factors dominantly expressed, resulting in rapid cell growth. Tropepe et al. (1999) also demonstrated that more spheres formed when E14.5 and E15.5 NSCs were cultured in medium modified with EGF and FGF. Moreover, since cells derived from E18 brain committed to differentiate to some extent, which led to the decrease in receptor numbers as well as proliferation capacity, less sphere were observed from these samples. Although E8.5 cells showed rapid proliferation and more spheres formed, contaminated fibroblast, which derived from mouse scalp, could excrete factors signaling cell adhesion and intense differentiation. In culture samples, many types of cell were observed, including, erythrocytes, fibroblasts, NSCs, some other cell types and lipid. However, those non-neural cells were eliminated after passages. Isolated candidate NSCs showed round-shape morphology, bright nucleus and sized between 8-12µm (Schumacher, 2003). Such cells grew in NSCs medium and formed cluster within 24 hours of culture. After 3-4 days, lipid and some other dead cells which could not grow in NSCs medium 10 aggregated into opaque clumps, in which several clusters of candidate cells (approximate 50µm) stuck. 100-150µm spheres were observed after 5-7 and reached the size of 250µm in day 10 of culture since cells in such sphere grew and proliferated, resulting in more daughter cells. Similar results also presented by Ge et al. (2012). (a) (c) (e) Figure 4.2’. Sphere formation from primary culture. (a) Isolated cells showed round-shape, red arrows: NSCs, blue arrows: erythrocytes (04/08/2013). (c) Opaque clumps after 4 days (04/04/2013). (e) Sphere after 11 days (04/19/2013). 3.1.2 Secondary culture After 2-3 days, cells were passed to other wells. Passage helped remove toxic metabolites, dead cells, provide nutrients and space for cell growth. When being mechanically dissociated, opaque clumps were removed and clusters of 3-4 cells were observed. Such clusters formed more small spheres within 24 hours and achieved clear round-shape after 3-4 days. Those spheres then continuously increased in size and had round, compact structure after passage 3 or 4. At the same time, other non-neural cells, which could not grow in NSC medium, died and 11 were removed, proving that during passages, only neural-like cells could survive and become dominant. After passage 7 or 8, cells in spheres died, leading to the lysis of protein linkages and the loss of spherical structure. The death of cells was shown to be related to caspase-3 (Milosevic et al., 2004), which is responsible for tumor suppression. 3.2 Determination of characteristics of candidate NSCs 3.2.1 Neurosphere assay Spheres of passage 4 were mechanically dissociated and cultured in 24-well plate. After 24 hours, small clusters formed and 50µm spheres were obtained in the next 2-3 days. Spheres became more compact and had the diameters ranging from 100200µm after 5-7 days and 250-400µm after passage 2-3. According to Reynolds and Weiss (1992), when single cells derived from SVZ were cultured in serum-free medium in the presence of EGF, they divided and initially adhered to the plate and detached to form spheres of proliferating cells after a few days. To assess self-renewal of cells, spheres were mechanically dissociated and cultured again in medium containing EGF to determine if single cells could form new spheres. Bez et al. (2003) indicated that 80% of cells derived from primary neurospheres could form secondary and tertiary neurospheres. However, low density of cultured cells leads to less forming spheres in comparison to high cell density since cell-cell interaction could induce excretion of growth factors (Pastrana et al., 2011). 12 3.2.2 Expression of markers of candidate NSCs 3.2.2.1 Sox1 expression of candidate NSCs Sox is a transcription factor in developing brains and helps maintain undifferentiated manner of NSCs by inhibition of neuronal differentiation. According Venere et al. (2012), Sox1 was an efficient marker for detecting NSCs derived from animal SGZ. Figure 4.6. Electrophoresis of RT-PCR products. 1: ladder, 2: GAPDH, 3: Sox1 (04/27/2013) As stated in Appendix 1, Sox1 gene sized approximately 165bp, which was similar to 170bp band observed in well 3, revealing the expression of Sox1 in these candidate cells. According to Bhaskar et al. (2005), Sox1 was expressed in most of E14 NSCs, and Chen et al. (2009) demonstrated that such amplified products of such gene in several animals ranged around 200bp. 13 The 300bp band on agarose gel was the housekeeping gene GAPDH, which was responsible for basic function of cells and popularly used in comparison of expression levels of different genes. 3.2.2.2 CD133 expression of candidate NSCs Firgure 4.7. 2D- dot plot Figure 4.8. Histogram (04/28/2013) (04/28/2013) Flow cytometry histogram revealed that 59.51% of candidate cell was CD133 positive. Integrated data of all above mentioned results indicated that such cells were NSCs (Figure 4.8). However, since cells were harvested after second passage, undesired cells were not completely eliminated, resulting in low proportion of CD133+ cells. In addition to NSCs, neurospheres consisted of glial cells and neural progenitors at different stages of mitosis and differentiation which affected flow cytometry results. 14 3.2.2.3 Nestin expression of candidate NSCs Stained spheres showed green fluorescence of FITC and blue fluorescence of Hoechst 33342, indicating the presence of cells with nuclei in neurosphere structure, and such cells were Nestin positive (Figure 4.9). In contrast, less cells located in outer layer expressed this protein. Nestin is a type of intermediate filaments in neural stem cell cytoskeleton; however, gene encoding for this protein is down regulated during cell differentiation, thus such protein was less expressed in larger sphere, which correlated with more differentiated cells. Due to compact structure of neurosphere, which might limit the access of labeled antibody, fluorescent emission of inner layers was not clearly observed. Figure 4.9. Nestin expression of NSCs (05/03/2013) 15
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