Summary of Biotechnology doctoral thesis: Evaluation of changes in proliferation and cytoskeletal structure of human umbilical cord mesenchymal stem cells under simulated microgravity

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The aim of this thesis was to gain an understanding of how the simulated microgravity condition affects the development of hucMSC by investigating the changes in cell morphology, proliferation and cytoskeleton structure of hucMSCs under in vitro condition.

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Nội dung Text: Summary of Biotechnology doctoral thesis: Evaluation of changes in proliferation and cytoskeletal structure of human umbilical cord mesenchymal stem cells under simulated microgravity

MINISTRY OF EDUCATION VIETNAM ACADEMY OF AND TRAINING SCIENCE AND TECHNOLOGY GRADUATE UNIVERSITY OF SCIENCE AND TECHNOLOGY ----------------------------- Hồ Nguyễn Quỳnh Chi EVALUATION OF CHANGES IN PROLIFERATION AND CYTOSKELETAL STRUCTURE OF HUMAN UMBILICAL CORD MESENCHYMAL STEM CELLS UNDER SIMULATED MICROGRAVITY Major: Biotechnology Code: 9 42 02 01 SUMMARY OF BIOTECHNOLOGY DOCTORAL THESIS Ho Chi Minh – 2021
The thesis was carried out at: Graduate University of Science and Technology - Vietnam Academy of Science and Technology Supervisor: Prof. Hoàng Nghĩa Sơn Reviewer 1: … Reviewer 2: … Reviewer 3: …. The thesis will be defended to the Academy-level doctoral thesis committee at Graduate University of Science and Technology - Vietnam Academy of Science and Technology at…, ……………… 2021. The thesis can be found at: - Library of Graduate University of Science and Technology - National Library of Vietnam
1 INTRODUCTION 1. Rationale In gravitational biology, studies of microgravity in space have shown many effects of gravity conditions on biological processes, gravity-sensitive mechanisms, or the gravity-based orientation of organisms. However, research in space has encountered a number of difficulties, including expensive research facilities and negative effects of microgravity on astronauts. To overcome the above difficulties, scientists have developed biological system models that simulate conditions similar to the microgravity in space for further gravitational studies on living organisms. Many studies showed that cells in different organisms behave differently in space than they do on Earth. Due to the great diversity of cell types in nature, the effects of microgravity on those cells are extremely diverse and often complex. Some studies on the role of microgravity in cell proliferation and differentiation have demonstrated that cells that grow in microgravity develop differently than under normal conditions, leading to notable changes in cell division. Cytoskeleton is also one of the most affected structure under microgravity. The cytoskeleton forms the main structure of the cell and includes interactions between microtubules, actin microfibers, intermediate microfibers and related proteins. Hence, the cytoskeleton is very much related to the cell shape. Abnormalities in the organization of microtubules and microfibers in the cytoskeleton can have a detrimental effect on the cell itself, even with very large consequences when the cell is in the embryonic stage. The mechanisms of proliferative and cytoskeletal structural changes under microgravity have not been clearly demonstrated yet.
2 Umbilical mesenchymal stem cells are potential cell lines, and their acquisition does not pose as many of the ethical issues as other strains of mesenchymal stem cells. Currently, there are not many reports on the effect of microgravity on umbilical medial stem cells, thus, the study used this cell line as the subject to evaluate the effects of microgravity. From the above reasons, this thesis research used 3D clinostat to create simulated microgravity environment to evaluate the effect of simulated microgravity conditions on the proliferation and cytoskeletal structure of human umbilical cord mesenchymal stem cells (hucMSCs). 2. Overall goals The aim of this thesis was to gain an understanding of how the simulated microgravity condition affects the development of hucMSC by investigating the changes in cell morphology, proliferation and cytoskeleton structure of hucMSCs under in vitro condition. 3. Objectives ‒ Evaluate the effect of simulated microgravity on the proliferation of hucMSCs. ‒ Evaluate the effect of simulated microgravity on the apoptosis of hucMSCs. ‒ Evaluate the effect of simulated microgravity on the morphology of nucleus and cytoplasm of hucMSCs. ‒ Evaluate the effect of simulated microgravity on the cytoskeleton of hucMSCs.
3 CHAPTER 1. LITERATURE REVIEW 1.1. The role of biological research in space This section outlines the importance and fields of biological research in space. 1.2. Introduction of microgravity and simulated microgravity This section introduces an overview and gives the concepts of simulated microgravity and microgravity. 1.3. Simulated microgravity systems This section introduces some simulated microgravity systems and the studies done on these systems. 1.4. Umbilical cord mesenchymal stem cells This section presents the biological properties of umbilical cord mesenchymal stem cells and their advantages in research. 1.5. Cell proliferation Presentation of the concepts of cell proliferation, cell cycle, factors that regulate the cell cycle, and studies on the effect of microgravity on cell proliferation. 1.6. Apoptosis This section presents the concept, factors that influence the apoptosis and methods to detect apoptosis. 1.7. Nucleus and cytoplasm This section presents the concepts, factors affecting the morphology of the cell nucleus and cytoplasm and methods to evaluate them. 1.8. Cytoskeleton This section presents the structural components of the cytoskeleton and studies on the effect of microgravity on the cytoskeleton.
4 CHAPTER 2. MATERIALS AND METHODS 2.1. Materials ‒ Human umbilical cord mesenchymal stem cells (hucMSCs) were provided by The Institute of Tropical Biology, Ho Chi Minh City. ‒ The 3D clinostat system creating the simulated microgravity environment was designed and manufactured in the project: “Evaluation of changes in the cytoskeleton structure in simulated microgravity condition”, from the Space Science and Technology program, code: VT-CB.15/18-20. 2.2. Methods 2.2.1. Cell culture 2.2.1.1. Thawing of frozen hucMSCs The cryotube of hucMSCs was added with 1 ml culture medium containing DMEM/Ham's F-12 with L-Glutamine (DMEM-12-A, Capricorn Scientific, Germany) supplemented with 15% FBS (FBS- HI-22B, Capricorn Scientific, Germany) and 1% Pen/Strep (15140- 122, Gibco, USA), then centrifuged at 1500 rpm for 5 minutes at room temperature to collect cell precipitate. The precipitate was re- suspended in the above culture medium and transferred to T25 flasks. The cells then were cultured at 37°C, 5% CO2. 2.2.1.2. Cell subculture hucMSCs were detached by Trypsin 0.25% (TRY-2B, Capricorn Scientific, Germany) and centrifuged at 1500 rpm for 5 minutes at room temperature to obtain cell precipitation. Cell precipitates were resuspended and cultured at 37°C, 5% CO2. 2.2.1.3. Cell cryopreservation hucMSCs were harvested and suspended with 500 μl DMEM/Ham's F-12 with L-Glutamine (DMEM-12-A, Capricorn
5 Scientific, Germany) and 500 μl HyCryo-STEM cryogenic medium (SR30002.02, GE Healthcare Life Sciences, USA). The cell suspension was transferred to a cryotube (430663, Corning, USA) and stored at -20°C for 1 hour, then stored overnight at -80°C and finally transferred to liquid nitrogen. 2.2.2. Evaluation of surface markers for human mesenchymal stem cells hucMSCs after thawing and culture were harvested and evaluated the stemness by Flow cytometry method using Human MSC analysis kit (562245, BD Biosciences, USA). Cell samples were analyzed by BD Accuri C6 flow cytometer system (BD Biosciences, USA). 2.2.3. Microgravity simulation hucMSCs were cultured in T-25 flasks (160430, Thermo Scientific, USA) and 96-well plates (161093, Thermo Scientific, USA) with densities of 1×105 cells/flask and 2×103 cells/well, respectively. The plates and cell flasks were filled with the culture medium. Cells were then divided into two groups: simulated microgravity group (SMG) and control group (Control). For the SMG group, cells were cultured in 3D-Clinostat machine system placed in a CO2 incubator and rotated at 1.3×10-3G. The control group was treated at 1G in the same CO2 incubator. Microgravity test conducted within 72 hours. 2.2.4. Evaluation of cell proliferation 2.2.4.1. Cell density hucMSCs were treated with WST-1 solution (11644807001, Roche, Switzerland) for 3.5 hours at 37ºC, 5% CO2. OD values per well were measured by GloMax® Explorer Multimode Microplate Reader spectrometer (Promega, USA) at 450 nm.
6 2.2.4.2. Cell cycle Flow cytometry: BD Accuri C6 flow cytometer (BD Biosciences, USA) was used to analyze changes in the cell cycle of hucMSCs. Transcript expression of cell cycle-regulated genes: The total RNA of hucMSCs was extracted with the ReliaPrepTM RNA Cell Miniprep System kit (Z6011, Promega, USA). Transcript expressions of CDK2, CDK6 and Cyclin A genes were evaluated by Realtime qRT-PCR method using 2x qPCR kit SyGreen 1-Step Go Hi-ROX kit (PB25.32.03, PCRBiosystem, UK). The thermal cycle was as follows: 45°C for 15 min, 95°C for 2 min, 40 cycles of 95°C for 10 sec and 62°C for 15 sec, 71 cycles of 60°C for 30 sec, 4°C for 30 min. Primer sequences include: CDK2 F: 5’- CCAGGAGTTACTTCTATGCCTGA-3’, R: 5’- TTCATCCAGGGGAGGTACAAC-3’; CDK6 F: 5’- TCTTCATTCACACCGAGTAGTGC-3’, R: 5’- TGAGGTTAGAGCCATCTGGAAA-3’; Cyclin A F: 5’- GCCATTAGTTTACCTGGACCCAGA-3’, R: 5’- CACTGACATGGAAGACAGGAACCT-3’; GAPDH F: 5’- CATGAGAAGTATGACAACAGCCT-3’, R: 5’- -∆∆Ct AGTCCTTTCCACGATACCAAAGT-3’. The Livak method 2 was applied to assess the relative expression level of gene expression. Translate expression of cell cycle-regulated genes: The expression level of Cyclin A1 + A2, CDK4, and CDK6 proteins was evaluated by Western Blot. GAPDH protein was used as a control. The antibodies used include: Anti-Cyclin A1 + Cyclin A2 antibody (ab185619, Abcam, USA), Anti-CDK4 antibody (ab137675, Abcam, USA), Anti-CDK6 antibody (ab124821, Abcam, USA), Anti-
7 GAPDH antibody (ab181602, Abcam, USA). ImageJ software (National Institutes of Health, Bethesda, USA) was used to measure the intensity of protein bands. 2.2.5. Evaluation of apoptosis 2.2.5.1. Cell harvesting hucMSCs were harvested as shown in Section 2.2.1.2. 2.2.5.2. Flow cytometry hucMSCs were treated with FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, USA) and analyzed by BD Accuri C6 flow cytometer system (BD Biosciences, USA). 2.2.5.3. Transcript expression of apoptosis related genes The transcript expression of Bax and Bcl-2 genes were evaluated by Realtime qRT-PCR method. The process of collecting total RNA and Realtime qRT-PCR reaction was performed as in Section 2.2.4.2. The thermal cycle was as follows: 45°C for 15 min, 95°C for 2 min, 40 cycles of 95°C for 10 sec and 52.2°C for 15 sec, 71 cycles of 60°C for 15 sec, 4°C for 30 min. The primer pairs include: Bax F: 5’-CCAGGAGTTACTTCTATGCCTGA-3’, R: 5’- TTCATCCAGGGGAGGTACAAC-3’; Bcl-2 F: 5’- TCTTCATTCACACCGAGTAGTGC-3’, R: 5’- TGAGGTTAGAGCCATCTGGAAA-3’; GAPDH F: 5’- CATGAGAAGTATGACAACAGCCT-3’, R: 5’- AGTCCTTTCCACGATACCAAAGT-3’. 2.2.6. Evaluation of the morphological changes in nucleus and cytoplasms The nucleus of hucMSCs were stained with Hoechst 33342 (14533, Sigma-Aldrich, USA) and observed under Cytell system (GE Healthcare, USA). The Cell Cycle App was used to evaluate
8 morphology, including the nuclear area, the nuclear intensity, and the nuclear shape value. ImageJ software (National Institutes of Health, Bethesda, USA) was used to evaluate cell area. The size of hucMSCs was also assessed by measuring FSC (Forward Scatter) index using flow cytometry method. 2.2.7. Evaluation of changes in cytoskeleton structure 2.2.7.1. Cell harvesting hucMSCs were harvested as shown in Section 2.2.1.2. 2.2.7.2. Transcript expression of genes encoding microtubles and microfilaments The transcript expression of α-tubulin 3 and β-actin genes were evaluated by Realtime qRT-PCR method. The process of collecting total RNA and Realtime qRT-PCR reaction was performed as in Section 2.2.4.2. The thermal cycle was as follows: 45°C for 15 min, 95°C for 2 min, 40 cycles of 95°C for 10 sec and 60°C for 15 sec, 71 cycles of 60°C for 15 sec, 4°C for 30 min. The primer pairs include: α-tubulin 3 F: 5’-CATTGAAAAGTTGTGGTCTGATCA-3’, R: 5’- GCTTGGGTCTGTAACAAAGCAT-3’; β-actin F: 5’- GAGCACAGAGCCTCGCCTTT-3’, R: 5’- AGAGGCGTACAGGGATAGCA-3’; GAPDH F: 5’- CATGAGAAGTATGACAACAGCCT-3’, R: 5’- AGTCCTTTCCACGATACCAAAGT-3’. 2.2.7.3. Translate expression of genes encoding microtubles and microfilaments Expression levels of α-tubulin and β-actin proteins were evaluated by Western Blot as in Section 2.2.4.2. The antibodies used include: Anti-beta Actin antibody (ab8226, Abcam, USA), Anti-alpha
9 Tubulin antibody (ab52866, Abcam, USA), Anti-GAPDH antibody (ab181602, Abcam, USA). 2.2.7.4. Evaluation of the reorganization of microtubles and microfilaments The microtubules were stained with SiR-Tubulin Kit (CY-SC002, Cytoskeleton, Inc., USA). The microfilaments were stained with Phalloidin CruzFluor ™ 488 Conjugate (sc-363791, Santa Cruz Biotechnology, USA). The cell nucleus were stained with Hoechst 33342 (14533, Sigma-Aldrich, USA). Cells were observed under the Cytell fluorescence microscope system (GE Healthcare, USA). The density of microfilament and microtubule bundles in hucMSCs was evaluated by measuring the fluorescence intensity of proteins in these bundles with ImageJ software (National Institutes of Health, Bethesda, USA). 2.2.8. Stastitical analysis Sigma Plot software (SYSTAT Software, USA) was used to analyze data in the study. One-way ANOVA method was used to evaluate differences between experimental groups, where p ≤ 0.05, p ≤ 0.01, p ≤ 0.001 were considered to be statistically significant difference. CHAPTER 3. RESULTS AND DISCUSSION 3.1. Evaluation of surface markers for human mesenchymal stem cells The expression of negative and positive markers for human mesenchymal stem cells is shown in Figure 3.1. The results of flow cytometry analysis showed that hucMSCs used in the study expressed positive with the human mesenchymal stem cell-specific markers CD90, CD73 and CD105 (Figure 3.1B2-B4) and negative
10 expression for these cell-line nonspecific markers in PE hMSC Negative Cocktail, including CD34, CD11B, CD19, CD45, and HLA-DR (Figure 3.1 B1). The negative for CD34, CD45, and CD19 markers indicates that the hucMSC population was not contaminated with hematopoietic progenitor cells, endothelial cells or white blood cells. Figure 3.1. Evaluation of surface markers in hucMSCs Furthermore, the negative expression of hucMSCs to CD19 markers showed that B cells and dendritic cells did not exist in the hucMSC cell population. In addition, negative expression of HAL- DR and CD11B markers indicate no white blood cells or macrophages in the hucMSC cell population. The above analysis results showed that the hucMSC cell population used in the study retains the characteristics of mesenchymal stem cells. 3.2. Evaluation of cell proliferation 3.2.1. Cell density Optical density of hucMSCs in two experimental groups was calculated and shown in Figure 3.2. Accordingly, the average optical
11 density in SMG group was 0.97 ± 0.05, significantly lower than the control group, which was 1.09 ± 0.13 (p ≤ 0.001). This result indicated that the proliferation of hucMSCs decreased when cultured in simulated microgravity condition. Figure 3.2. The optical density of hucMSCs evaluated by WST-1 assay. A: Optical density chart of two experimental groups. B: hucMSCs under normal condition. C: hucMSCs under SMG condition. ***: p ≤ 0.001. Scale bar: 111.82 μm 3.2.2. Cell cycle 3.2.2.1. Cell ratio in each phase of the cell cycle The results of cell cycle analysis in Figure 3.3 show that the proportion of hucMSCs entering the resting phase G0/G1 in the SMG group was 85.65 ± 0.65%, significantly higher than that in the control group, which was 72.27 ± 1.28%. In addition, the proportion of hucMSCs entering the G2/M phase in the SMG group also decreased compared with the control group (5.65 ± 0.05% in the SMG group and 22.10 ± 1.27% in the control group). These differences were statistically significant when analyzed by One-way ANOVA with p ≤ 0.01. This data shows that simulated microgravity can reduce the division and tends to induce the arrest phase of hucMSCs.
12 Figure 3.3. Cell cycle analysis of hucMSCs by flow cytometry 3.2.2.2. Transcript expression of cell cycle-regulated genes mRNA expression of CDK2, CDK6 and Cyclin A in hucMSCs decreased with statistical significance when cultured in simulated microgravity condition (Figure 3.4). CDK2 in the SMG group showed a strong decline to 0.59 ± 0.07 compared to the control group (1.01 ± 0.13) (p ≤ 0.001). CDK6 reduced lighter when the expression level in SMG group was 0.68 ± 0.05 compared with the control (1.00 ± 0.14) (p ≤ 0.01). Cyclin A had the slightest decrease with expression levels in the SMG group was 0.83 ± 0.09 and in the control group was 1.00 ± 0.09 (p ≤ 0.05). Figure 3.4. Transcript expression of cell cycle-regulated genes. ***: p ≤ 0.001, *: p ≤ 0.05 3.2.2.3. Translate expression of cell cycle-regulated genes The expression of Cyclin A1+A2, CDK4 and CDK6 proteins in hucMSC tended to decrease when cultured in simulated microgravity
13 condition (Figure 3.5). Cyclin A1+A2 had the most obvious reduction when the expression level in the SMG group was 0.34 ± 0.04, nearly one third of the expression level in the control group (1. 00 ± 0.07) (p ≤ 0.001). CDK4 and CDK6 expression levels in the SMG group were 0.79 ± 0.01 and 0.82 ± 0.01, respectively, slightly decrease compared to the control group (1.00 ± 0.06 in CDK4 and 1.00 ± 0.10 in CDK6). Figure 3.5. Translate expression of cell cycle-regulated genes. ***: p ≤ 0.001, **: p ≤ 0.01, *: p ≤ 0.05 3.3. Evaluation of apoptosis 3.3.1. Apoptosis ratio Flow cytometry analysis showed that there was no significant difference in the survival of hucMSCs from the control group (93.20 ± 0.35%) and SMG group (92.85 ± 1.15%) (Figure 3.6). The total proportion of apoptosis (early apoptosis and late apoptosis) of hucMSCs from the SMG group was similar to the control. Figure 3.6. Viability and apoptosis ratios of hucMSCs
3.3.2. Transcript expression of apoptosis related genes Realtime qRT-PCR result from Figure 3.7 showed that there was no significant difference in mRNA expression of Bcl-2 and Bax in hucMSCs between the control group and SMG group. This suggests that apoptosis did not lead to a decrease in proliferation of hucMSCs. Figure 3.7. Transcript expression of apoptosis related genes 3.4. Evaluation of the morphological changes in nucleus and cytoplasms 3.4.1. Nuclear morphology Nuclear morphology of hucMSCs underwent some changes when they were cultured in simulated microgravity. In Figure 3.8, the nuclear intensity of hucMSCs decreased in the simulated microgravity environment, as the intensity in the SMG group was 5629317 ± 39469 compared to the control group (5957254 ± 65063) (p ≤ 0.001). Meanwhile, the nuclear area of hucMSCs did not change significantly when there was no significant difference between the SMG group (314.04 ± 2.55 µm2) and the control group (318.07 ± 1.73 µm2) (Figure 3.9). The nuclear shape value represents the level of symmetry of the cell nucleus, the more symmetrical the nucleus, the closer the value will approach 1.0. Figure 3.10 shows that the nuclear shape values of hucMSCs in the SMG group (0.89 ± 0.002) and the control group (0.88 ± 0.003) were not significant different.
15 The distribution of hucMSCs according to nuclear shape and nuclear intensity between the two experimental groups was quite similar (Figures 3.11 and 3.12). The control group had nuclear intensity distribution over the threshold of 1.5 × 107, while most of the nuclear intensity values of the SMG group were below the threshold of 1.5 × 107. Figure 3.8. Nuclear Figure 3.9. Nuclear Figure 3.10. Nuclear intensity of hucMSCs. area of hucMSCs shape of hucMSCs ***: p ≤ 0.001 Figure 3.11. Nuclear Figure 3.12. Nuclear distribution in the relationship distribution in the relationship between nuclear shape and between nuclear area and nuclear intensity in hucMSCs nuclear intensity in hucMSCs
16 3.4.2. Cytoplasmic morphology Figure 3.13A and 3.13B show the morphology of hucMSCs in SMG and control groups, which have characteristic elongated forms of mesenchymal stem cells. However, the cytoplasm of the hucMSCs in the SMG group tended to be much more extensive than in the control group. This statement is supplemented by the results of cell area Figure 3.13. Cytoplasmic morphology measurement in Figure of hucMSCs. A, B: Cytoplasmic 3.13C, in which the morphology of hucMSCs under normal mean area of hucMSCs conditions (A) and simulated in the SMG group was microgravity (B). C: Histogram of cell 14083.34 ± 1069.38 area assessment in hucMSCs. *: p ≤ µm2, higher than the 0.05. Scale bar: 223.64 µm control group. (11368.67 ± 535.27 µm2) (p ≤ 0.05). The hucMSCs also showed normal growth in both groups when no signs of apoptotic bodies were detected in the populations. The results in Figure 3.14 show that the FSC value of hucMSCs in the SMG group increased with the mean value of 10803988.58 ± 20960.12, higher than the control group (9829898.02 ± 206370.30) (p ≤ 0.01). This shows that simulated microgravity condition increased cell size in hucMSCs.
17 Figure 3.14. FSC value indicates cell size in hucMSCs. A, B: Graph showing FSC values of hucMSC in the control group (A) and SMG group (B). C: Chart of FSC mean values. **: p ≤ 0.01 The results of cytoplasmological analysis showed that simulated microgravity condition caused morphological changes of hucMSCs after 3 days of culture. Specifically, hucMSCs in simulated microgravity tend to expand their area than under normal condition. 3.5. Evaluation of changes in cytoskeleton structure 3.5.1. Transcript expression of genes encoding microtubles and microfilaments Realtime qRT-PCR results showed that α-tubulin 3 and β-actin decreased mRNA expression level when hucMSCs were cultured in SMG condition (Figure 3.15). Figure 3.15. Transcript expression of genes encoding microtubles and microfilaments. ***: p ≤ 0.001
18 The expression level of β-actin in SMG group was 0.60 ± 0.03, much lower than the control group, which was 1.00 ± 0.06 (p ≤ 0.001). The change of α-tubulin 3 was even more pronounced when the expression level in the SMG group was 0.23 ± 0.03 decreased by nearly five times compared to the control group (1.00 ± 0.08) (p ≤ 0.001). 3.5.2. Translate expression of genes encoding microtubles and microfilaments The protein expression of β-actin in microfilaments and α-tubulin in microtubules is shown in Figure 3.16. Accordingly, the β-actin protein strongly declined in simulated microgravity condition. The expression level of β-actin in the SMG group was 0.06 ± 0.01, reduced 16 times compared to the control group (1.00 ± 0.02). Protein expression of α-tubulin also decreased when the expression level in the SMG group was 0.42 ± 0.01 and in the control group was 1.00 ± 0.02. These differences are considered as statistically significant by One-way ANOVA analysis (p ≤ 0.001). Figure 3.16. Translate expression of genes encoding microtubles and microfilaments. ***: p ≤ 0.001 3.5.3. The reorganization of microtubles and microfilaments