Nội dung bản trích yếu Bệnh Parkinson là bệnh thoái hóa thần kinh tiến triển thường gặp ở những người trên 65 tuổi, với tỷ lệ mắc bệnh ở Việt Nam khoảng 1,6%. Phương pháp điều trị đầu tay là sử dụng các thuốc tiền chuyển hóa của dopamin như levodopa. Tuy nhiên điều trị nội khoa bằng thuốc thường người bệnh chỉ đáp ứng tốt trong 5 – 10 năm đầu do gặp phải hiện tượng kháng thuốc điều trị. Vì vậy, đây là nghiên cứu nhằm tiếp cận hướng điều trị mới đó là sử dụng tế bào gốc có khả năng biệt hóa thành nơron tiết dopamin để thay thế các nơron bị thoái hóa. Nghiên cứu được tiến hành trên đối tượng là 776 phôi chuột cống trắng từ 10,5 đến 14,5 ngày tuổi và 201 phôi – thai người từ 6 đến 8 tuần tuổi với chất liệu nghiên cứu là mẫu tế bào gốc ngoại bì thần kinh của các phôi đó nhằm hướng đến hai mục tiêu chính là (1) Phân lập, tăng sinh, biệt hóa và bảo quản lạnh tế bào gốc ngoại bì thần kinh phôi chuột cống trắng thành tế bào dạng tiết dopamin; (2) Phân lập, tăng sinh và biệt hóa tế bào gốc ngoại bì thần kinh phôi người thành tế bào dạng tiết dopamin. Thiết kế nghiên cứu thực hiện qua hai giai đoạn, giai đoạn 1 thực nghiệm trên phôi chuột cống trắng và giai đoạn 2 thực nghiệm trên phôi người. Mô hình nghiên cứu được thiết kế gồm các bước: thu thập phôi, phân lập mẫu mô sàn não giữa, tạo dịch treo tế bào gốc. Sau khi tạo được dịch treo tế bào, một phần được nuôi cấy tăng sinh tạo nơron tiết dopamin. Sử dụng môi trường nuôi cấy tế bào gốc thần kinh (M-NECS): môi trường nuôi cấy cơ bản DMEM/F12 tỷ lệ 1:1 có bổ sung thêm các yếu tố: Fetal Bovine Serum (FBS) 10%; Epithelial Grow Factor (EGF) 20ng/ml và basic Fibroblast Grow Factor – 2 (bFGF-2) (20ng/ml), (invitrogen, Mỹ); Penicillin - Streptomycin (100IU/ml) (Thermo Scientific, Mỹ); Amphotericine B (0,25µg/ml); Progesterone (20nM), Putrescine (62nM), muối Selenit (30nM), (Sigma, Mỹ); Insulin – Transferine – Selenium (25ng/ml) (Gibco, Mỹ). Các tế bào sau nuôi cấy được thu hoạch và định danh bằng các phương pháp mô học như nhuộm Hematoxyline – Eosine, nhuộm Giemsa để đánh giá hình thái tế bào, nhuộm Cajal II để đánh giá sự hiện diện của các xơ thần kinh trong tế bào, nhuộm Hóa mô miễn dịch với marker đặc hiệu cho nơron tiết dopamin là Vimentin và Tyroxin Hydroxylase (TH), đếm mật độ tế bào bắt màu với marker TH để xác định tỷ lệ nơron tiết dopamin trong mẫu tế bào thu hoạch được. Một phần khác sẽ được bảo quản lạnh trong môi trường cơ bản DMEM có bổ sung 10% Dimethyl Sulfoxide (DMSO) sau đó đánh giá tỷ lệ sống của tế bào sau rã đông cũng như khả năng nuôi cấy của tế bào sau bảo quản lạnh. Sau quá trình nghiên cứu, chúng tôi thu được một số kết quả sau: Đối với mục tiêu (1), các kết quả nghiên cứu cho thấy khả năng phân lập và nuôi cấy mẫu tế bào gốc thần kinh phôi chuột tốt nhất ở tuổi phôi 12,5 đến 13,5 ngày tuổi. Tỷ lệ phân lập thành công 100% ở nhóm tuổi phôi này với số lượng tế bào thu được khoảng 1,34 ± 0,048 x 105 tế bào/ml. Các tế bào sau nuôi cấy có đặc điểm của tế bào của mô thần kinh với nhiều tế bào hình sao với các nhánh bào tương tỏa ra xung quanh liên hệ với các tế bào gần kề. Nhuộm Cajal II thấy hình ảnh các xơ thần kinh trong tế bào bắt màu nâu vàng rõ. Sử dụng marker TH để đánh dấu nơron tiết dopamin, quan sát thấy nhiều tế bào dương tính với TH trong mẫu nuôi cấy. Tỷ lệ nơron dương tính với TH khoảng 5,38 ± 3,56%. Bảo quản lạnh các tế bào gốc thần kinh phôi sau phân lập trong môi trường cho tỷ lệ sống 64,9%. Đối với mục tiêu (2), việc phân lập, tăng sinh và biệt hóa tế bào gốc thần kinh phôi – thai người để tạo nơron tiết dopamin có thể thực hiện được bằng nguồn phôi được lấy từ kỹ thuật giảm thiểu thai trong điều trị hỗ trợ sinh sản. Tuổi phôi thích hợp cho phân lập và nuôi cấy từ 6,5 đến 7,5 tuần tuổi. Các tế bào sau nuôi cấy cũng là các tế bào của mô thần kinh và dương tính với marker TH – thể hiện sự tồn tại của nơron tiết dopamin trong mẫu thu hoạch được. Đây là một đề tài nghiên cứu rất mới. Tuy có được thành công bước đầu đã nuôi tạo được các nơron tiết dopamin nhưng vẫn còn vài khó khăn đặc biệt trong việc nghiên cứu trên phôi người. Như việc khó khăn trong phân lập các phôi sau giảm thiểu do đặc điểm không toàn vẹn của phôi. Vì vậy cần thêm những nghiên cứu trong tương lai để hoàn thiện quy trình nuôi cấy, tăng tỷ lệ tế bào tiết dopamin sau nuôi cấy cũng như tăng tỷ lệ tế bào sống sau bảo quản lạnh tế bào gốc ngoại bì thần kinh để có thể sẵn sàng nguồn tế bào tiến tới ứng dụng thử nghiệm trên động vật và xa hơn là điều trị trên lâm sàng.
1 INTRODUCTION The question Nowadays, the study and application of insights at the cellular and subcellular levels are at the forefront of modern medicine One of those key areas is the research, understand and use of stem cells in such treatment as transplantation of tissue and cells into the human body to restore part or all of the function of tissues and cells after pathological injury or aging, after mechanical trauma or due to congenital defects The idea of a stem cell transplant to treat Parkinson's disease, a progressive degenerative neurological disorder caused by decreased function of dopamine-secreting neurons in the brain, was started in the 1980s Around the world, many animal and human studies have shown that stem cell transplantation significantly improves symptoms of Parkinson's disease Various types of stem cells have been tested in animals: bone marrow stem cells, induced stem cells (iPS cells), fetal neural ectoderm stem cells (mesencephalic neuroepithelial stem cells = M-NECs) Among the above types, human fetal neural ectoderm stem cells have been clinically tested in humans and have yielded positive results because the cells here have a high rate of differentiation into posttransplant dopamine-secreting neurons The graft tissue is in good agreement with the host tissue and there is no evidence of abnormal dopamine secretion from the graft on the host tissue In Vietnam, the rate of Parkinson's disease is about 1,6% in people over 65 years old, and tends to increase In addition, medical treatment using drugs that are metabolites of dopamine such as Levodopa is only effective for about 5-10 years due to drug resistance With the desire to approach a new method in the treatment of Parkinson's disease as well as the desire to improve the quality of life for patients, we conducted a study on the topic: "Isolation, proliferation and differentiation of foreign embryonic neural dermis stem cells – into the dopamine-secreting cell” with the goal of: Isolation, proliferation, differentiation and cryopreservation of rat embryonic neural ectoderm stem cells into dopamine-secreting cells Initially isolated, proliferated and differentiated human embryonic neural ectoderm stem cells into dopamine-secreting cells 2 What are the new insights from the thesis The thesis has succeeded in isolating, proliferation, differentiation and cryopreservation of white mice embryonic neural ectoderm stem cells into dopamine-secreting cells Initial success in isolating, proliferating and differentiating human embryonic neural stem cells into dopamine-secreting cells This is a large-scale, elaborate study on isolating, culturing and differentiating mice and human neural stem cells, serving as a premise for experimental studies on the treatment of Parkinson's disease by neural ectoderm root stem cells in animals as well as application in future volunteer patients Thesis structure The thesis consists of 115 pages with pages of Problem Statement, 33 pages of Overview, 17 pages of Research Objects and Methods, 30 pages of Results, 30 pages of Discussion, page of Conclusion, page of Recommendations and Research Direction new The thesis has tables of data, charts, 27 images of research cells; 115 References, appendices, list of mice embryos, list of patients Chapter OVERVIEW 1.1 Neural stem cells: definition and characteristics Neural stem cells are cells that have the ability to proliferate and selfrenew while remaining undifferentiated throughout their lifespan Selfrenewal is possible through both symmetric and asymmetric division While symmetric division produces two stem cells that increase the stem cell population, asymmetric division produces one stem cell and the other begins the process of differentiation Thus, neural stem cells are undifferentiated cells with the ability to self-renew as well as the ability to differentiate into a committed cell line Neural stem cells have the ability to differentiate into many different types of cells such as neurons, astrocytes or oligodendrocytes 1.2 The process of formation and differentiation of dopaminesecreting neurons in vivo Stem cells responsible for differentiation into dopamine-secreting neurons reside in the midbrain floor of the embryo During embryonic development, in the embryonic stage, when the neural tube is newly closed, the neural tube wall is an epithelium consisting of a single row of cells called the neural epithelium 3 During development, the midbrain wall thickens due to the intense proliferation of these epithelial cells The cells in the tubular wall initially have the form of pseudostratified columnar epithelium, the cytoplasm is spread throughout the wall thickness, and the nuclei are distributed to varying degrees of elevation In the deep layer of the tubular wall, the cells divide strongly, forming the reproductive layer in this region In the deep layer of the tubular wall, the cells divide strongly, forming the reproductive layer in this region After being formed in the reproductive layer, the cells migrate to the periphery of the tubule wall and begin the process of differentiation At an early stage, dopaminesecreting progenitor cells express markers Shh, Lmx1a, Lmx1b in the cytoplasm The progenitor cells then further differentiate into immature dopamine-secreting neurons These neurons begin to leave the reproductive region along the branches of the radial neurons to enter the medial region in the tubular wall During migration, dopamine-secreting neuroblasts continue to differentiate to become mature dopaminesecreting cells At this stage, cells appear to express the marker Thyroxine Hydroxylase (TH) Thus, dopamine-secreting progenitor cells begin to form in the floor of the midbrain, then undergo a series of development, differentiation and migration steps to certain locations such as: black sickle, hypothalamus, olfactory bulb, retina In terms of time, this process starts from day in mice and week after fertilization in humans It is these insights that have greatly helped in the search for optimal cell lines for the treatment of Parkinson's disease 1.2 Mid-embryonic brain stem cells and therapeutic applications Using a piece of midbrain floor tissue: Authors Perlow et al (1979) carried out grafting of tissue pieces isolated from the midbrain floor into the striatum of mice striatum that had been induced with 6Hydroxydopamine (6-OHDA), the results showed Clinical improvement was observed in some post-transplant mice However, this technique revealed many limitations such as: low survival rate of mice after transplantation, symptom improvement rate in heterogeneous study batches These limitations may be related to poor nutrition because the cells in the graft not come into contact with the blood vessels and cerebrospinal fluid of the host tissue 4 To overcome the limitations of using a piece of midbrain floor tissue, many authors have used cell suspension isolated from this tissue fragment to inject into the striatum of mice with Parkinson's disease The results were very positive when the cure rate of the mice after transplantation was stable between the study lots Moreover, when observing the post-transplant striatum structure by immunohistochemistry, the authors found the existence of embryonic cells in the host These cells live and create neurons associated with host tissue cells, which can be observed neuronal migration up to 0.5mm - 1mm from the injection site The successes in experimental mice prompted scientists to conduct tests on volunteer patients A sample of stem cell suspension was injected into the brain of a Parkinson's patient Post-transplantation recorded improvement in symptoms of movement disorders as well as symptoms of insomnia in Parkinson's patients A large controlled clinical trial of 34 volunteers who received an embryonic neural stem cell suspension transplant and a placebo group by Olanow et al in 1992 also showed improvement in symptoms of the treated patients compared with the untreated group, especially in patients with mild Parkinson's disease Interestingly, the authors found that there was a large difference in improvement between the 4-embryo group compared with the 1-embryo group The authors therefore found a link between the number of cells used and the ability to improve symptoms While the source of allogeneic embryonic tissue is very small, so to increase the number of transplanted cells, scientists have had the idea of culturing these cell mixtures to increase the amount of dopaminesecreting neurons, thus help optimize the effectiveness of treatment 1.4 Isolation, culture and cryopreservation of embryonic midbrain floor stem cells 1.4.1 Isolation of neural stem cells Isolation of neural stem cells includes the following steps: tissue extraction, cell division, and cell population homogenization In order to extract, it is necessary to first have a complete knowledge of the neural stem cell foci location and localization techniques to obtain reliable tissue samples After obtaining a brain tissue sample, special attention should be paid to preservation, which should be done within a few hours because the survival rate of cells decreases over time After extracting the tissue piece in the right position, proceed to separate the cells that produce the suspension Either enzymatic or mechanical cell separation can be used The enzyme commonly used to separate cells is trypsin In particular, trypsin is often used in combination with ethylenediamine tetra acid (EDTA), which is a substance that binds to Ca2 + to increase the effect of cutting bonds between cells Normally, enzymatic cell separation is not sufficient to completely isolate neural stem cells in tissue Therefore, scientists often coordinate with mechanical cell separation by using a pasteur pipette that sucks up and down many times After the separation process, the cells by enzymes and mechanics, the resulting cell population will be homogenized by filtering through a filter with a pore size of 70 µm or 100 µm It is also possible to rely on fluorescence signals with specific markers to select neural stem cells 1.4.2 Culture of neural stem cells The medium commonly used for culturing neural stem cells is the basal medium supplemented with growth factors The commonly used base medium is DMEM which is made up of a mixture of inorganic salts, amino acids and vitamins DMEM is often combined with a 1:1 ratio of Ham's F12 solution to increase the level of nutrients and various inorganic salts In addition, the basal medium also needs to be supplemented with other growth factors such as: epithelial growth factor (EGF), fibroblast growth factor (FGF), insulin, transferrin or putrescine, serum, antibiotics, antifungal These factors are often added when cultured because they are only used for a short time at 4℃ These factors, when combined in a culture medium, help neural stem cells both retain the "original" nature of the cells, prolong the time of cell proliferation, and increase efficiency in differentiation into dopamine-secreting neurons Culture methods: There are two main methods of culturing neural stem cells, namely suspension cluster culture and single layer culture method In which, the most popular method is culturing the suspension of nerve cells Accordingly, the nerve cells will be grown in a medium without binders but full of growth factors This allows the cells to grow and form clusters of cells Some researchers suggest that clusters of cells are similar to a microenvironment that stimulates the growth of neurons because they have direct cell-cell interactions within the clusters However, this method also has some disadvantages such as when the clusters are large, the diffusion of nutrients inward is more difficult or the high concentration of cells in the clusters will limit the evaluation 6 individual cells as well as hindering the study of cell differentiation Single layer neural stem cell culture is the use of adhesion agents on the surface of the culture plate to stimulate neuronal cell line adhesion and proliferation, while being unfavorable for other cell lines to grow This method seems to overcome the limitations of cluster culture such as better penetration of nutrients into cells or easier study of single cells However, the ability to proliferate and differentiate into mature neurons is not as effective as cluster culture The reason given by the authors is the advantage of the microenvironment created by the cell clusters, which have mutual effects in the proliferation and differentiation of neural stem cells The neural stem cells after the culture process will be harvested and used identification measures to identify the cells of the neural tissue as well as the dopamine-secreting neurons The commonly used measures are morphological assessment and specific markers TH is commonly used to assess the presence of dopamine-secreting neurons 1.4.3 Cryopreservation of midbrain stem cells Cryopreservation is the storage of cells at low temperatures while preserving cell structure and viability The neurons in the sample after isolation or culture have some special characteristics such as: many cells at different stages of differentiation, including both single cells and cell clusters Therefore, the selection of medium and cryopreservation method to optimize cell recovery has also been studied by many authors Using membrane-permeable cryoprotectant Dimethyl sulfoxide (DMSO) at a concentration of 7-10% gave the highest survival rate after thawing 7 Chapter RESEARCH SUBJECTS AND METHODS 2.1 Research object and location 2.1.1 Research subjects Research was conducted on: - There are 776 Wistar purebred white mice embryos from 10,5 to 14,5 days old Among those 186 embryos were used to study the structure of midbrain and 590 embryos were used for cell isolation and culture - 201 human embryos - human fetuses from 6,5 to weeks old without infectious diseases such as HIV, Hepatitis B, Syphilis Embryos were collected by the method of pregnancy reduction in women with multiple pregnancies during Assisted Reproductive Technology treatment and with the consent of the pregnant woman 2.1.2 Place and time of study The study was conducted at the Cell Culture Lab, Department of Tissue and Embryology, Hanoi Medical University from October 2014 to October 2018 2.2 Research Methods The research was conducted experimentally in the lab and divided into phases: 2.2.1 Steps to take on mice embryos Diagram depicting the experimental steps on mice embryos: Figure 2.1 Study diagram of cultured embryonic neural ectoderm stem cell in mice 2.2.2 Steps on Human Embryos Embryos – human fetuses – weeks old were collected from fetuses undergoing pregnancy reduction procedures in women with multiple pregnancies during Assisted Reproductive Therapy After being collected from the hospital, the embryos were placed in Falcon tubes containing 10ml of HBSS medium, stored in a refrigerator (temperature -4℃) then transferred to cell culture lab within hour The embryos that meet the criteria for research will continue the same process of isolation, culture and identification as above 2.2.3 Research target * For subject no 1: Microscopic morphology, virus, and immunohistochemistry of embryonic middle brain samples according to embryonic age in mice Microscopic morphology, virus, and immunohistochemistry of mice embryonic cells after culture The percentage of neurons secreting dopamine – TH (+) after culture The percentage of cell clusters with dopamine - TH (+) neurons after culture Cell survival rate after cryopreservation with liquid nitrogen *For subject no 2: Microscopic morphology, virology, immunohistochemistry of human embryonic midbrain samples Microscopic morphology, virus, immunohistochemistry of human embryonic cells after culture The percentage of neurons secreting dopamine – TH (+) after culture The percentage of cell clusters with dopamine - TH (+) neurons after culture 2.2.4 Techniques used in the study a Microscopy techniques to evaluate the microscopic morphology of cells: Hematoxylin – Eosin (HE) staining to study the microstructure of the brain between mice and human embryos by blue/red staining with Hematoxylin and Eosin dyes Giemsa stain for surface visualization of cultured cells using 10% Giemsa dye mixed with distilled water immediately before use Golgi II staining technique was intended to determine the presence of neurofibromatosis in the midbrain tissue of the mice embryo as well as in cell cultures Use 1,5% AgNO3 solution 9 Stained slides will be read under an optical microscope using objective lenses with magnifications of x10, x20 and x40 b Electron microscopy techniques Scanning electron microscopy (SEM) is intended to observe the surface microstructure of cells after culture Penetration electron microscopy (TEM) aims to observe the superstructure of the cell membrane, cytoplasm, nucleus, organelles and intercellular relationships The sample after making the slide will be read by scanning electron microscope and penetrating electron microscope c Immunohistochemistry Immunohistochemical staining with Vimentin marker (ab92745USA) to observe the presence of Vimentin fibers in embryonic brain cells as well as post cultured cells to identify neural stem cells and Tyrosine Hydroxylase (TH) marker (ab112 - USA) to observe the presence of Tyrosine Hydroxylase enzyme in embryonic brain cells, cultured cells to identify dopamine-secreting cells The slides after staining will be observed under a fluorescence microscope d TH (+) cell counting technique Postharvest cells were stained immunohistochemically with the TH marker Using fluorescence microscopy count the red-spotted cells in the microfield – that is, TH(+) cells Randomly move 10 microfields under x20 magnification Count the total number of cells in the observed microfields Ratio of TH(+) cells = Number of TH(+) cells/Total number of cells counted 2.2.6 Research ethics This study is part of the content of the State Independent Project approved by the Ethical Council of Hanoi Medical University in accordance with Official Letter No 148/HĐĐĐHYHN dated August 6, 2014 and approved by the Research Ethics Committee The study had the consent of the grassroots leadership and the voluntary consent of the pregnant women undergoing the procedure to reduce pregnancy, with a commitment of consent Patient information is kept confidential for research purposes only The list of patients will not disclose their full names to ensure confidentiality as required by law The research is only for the patient's wellbeing and has no other purpose 10 Chapter RESEARCH RESULTS 3.1 Isolation, proliferation, differentiation and cryopreservation of embryonic midbrain neural ectodermal stem cells in mice 3.1.1 Isolation and proliferative culture of mice embryonic midbrain ectodermal stem cells according to embryo age group In the first stage, 148 mice embryos from 10,5 to 14,5 days old (E10,5 – E14,5) were used to evaluate the ability to isolate and culture midbrain ectodermal stem cells based on embryo age Table 3.1 The rate of successful extraction of midbrain tissue samples by embryo age: Embryo age E10,5 E11,5 E12,5 E13,5 E14,5 Total (n) Number of embryos 30 25 30 33 30 148 Number of embryos successfully extracted 19 18 27 30 27 121 Rate (%) 63,3 72 90 90,9 90 81,7 Table 3.1 shows the rate of successful extraction of the midbrain by age groups of embryos E10,5; E11,5; E12,5; E13,5; E14,5 respectively: 63,3%; 72%; 90%; 90,9% and 90% For 10,5-day-old embryos, the success rate of midbrain extraction was the lowest At higher embryonic age, the rate of successful extraction was also higher and was highest in the age group E12,5 – E14,5 b Total number of cells after isolation After extraction, the midbrain tissue sample was incubated with Dispase and Trypsin -EDTA enzymes to create a sample of cell suspension To evaluate the efficiency of midbrain floor stem cell isolation, we counted the total number of cells in the suspension sample The average number of cells according to embryo age is shown in the following chart 3.1: 11 Chart 3.1 Total number of cells after isolation Total number of cells after isolation was lowest at embryo age 10,5 days (approximately 0,78 ± 0,05 x 10^5 cells) and highest at embryo age 14,5 days (approximately 1,4 ± 0,51 x 10^5 cells) There are differences in cell number between 11,5 and 12,5 days old embryos (about 0.82 ± 0,085 x 10^5 compared to 1,34 ± 0,048 TB x 10^5 cells) c Growth rate of cultured samples according to embryo age After isolating suspension samples, neural ectodermal stem cells were cultured in M-NECS medium to evaluate the proliferative efficiency of the samples The results of cell proliferation are shown in Table 3.2: Table 3.2 Sample growth rate according to embryo age: Embryo age E10,5 E11,5 E12,5 E13,5 E14,5 19 18 27 30 27 No of sample 16 27 30 27 No of growth Rate 10,5% 88,9% 100% 100% 100% Table 3.2 showed a very low growth rate (10.5%) in E10,5 rat embryos while embryos at E11,5 stage had a higher growth rate (88.9%) Embryos E12,5 – E14,5 have the best sample growth rate of 100% 3.1.2 Proliferation and differentiation of mice embryonic neural ectoderm stem cells E12,5 - E13,5 generates dopamine-secreting neurons After selecting the appropriate embryo age for culturing dopaminesecreting neurons, it was found that the most suitable embryo age for midbrain stem cell proliferation culture was the embryonic stage of 12,5 – 13,5 days old We conducted experiments on 442 samples of midbrain tissue extracted from white rat embryos stage E12,5 – 13,5 Of which, 370 samples were cultured to proliferate dopamine-secreting neurons, the 12 remaining 72 samples were isolated into cell suspension for cryopreservation Results obtained 415 cell wells after culture Of which 138 samples were stained with HE, Giemsa and Golgi II; 159 samples were stained Immunohistochemically with markers TH and Vimentin; 40 samples were prepared using Scanning Electron Microscopy (SEM) and Penetration Electron Microscopy (TEM) slides a Development of cultured cell over time Two days after culture: most of the cells were adherent, beginning to observe the neuron-like image: astrocytes, with a few short cytoplasmic branches radiating from the stem, ending with bulging tips Some cells have long branches that come into contact with adjacent cells (Figure 3.1A) Subsequent days: cells proliferate rapidly, with many epithelial cells forming clusters, scattered with scattered astrocytes, many cells with long branches like neurons that are scattered or formed cluster of neurons, the cell branches are long and interlaced (Figure 3.1B, C, D) Cells in neuron-like clusters tend to spread to the surrounding area and at the same time radiate many cytoplasmic branches associated with adjacent clusters (Figure 3.1C, D): The average time for the culture samples to grow to the bottom of the cage ranged from 7,4 ± 2.3 days Culture time and cell growth rate were fairly uniform in the samples Figure 3.1.Cultivated midbrain stem cells A After days (reverse bronchoscopy x100) B After days (reverse bronchoscopy x100) C After days (reverse bronchoscopy x100) D After days (reverse bronchoscopy x100) 13 b Identification of cells after culture - Microscopic form Figure 3.2 Microscopic morphology of cultured midbrain stem cells Midbrain stem cells after days of culture (Giemsa x100) Midbrain stem cells after days of culture (Giemsa x 200) Midbrain stem cells after days of culture (Cajal II x 100) - Super microscopic form Using scanning electron microscopy to observe the surface of cultured cells, see images of neurons with large, spherical or rhomboid bodies, long cytoplasmic branches connecting with the cytoplasmic branches of adjacent cells (Figure 3.3): 14 Figure 3.3 Midbrain stem cells after days of culture (SEM) On cross-sectional slices of cultured cells, we found many images of cytoplasmic branches that cut across the cell (Figure 3.4A) There are cytoplasmic dendrites that enlarge into growth cones (Figure 3.4B) and also synapses formed between cytoplasmic branches (Figure 3.4C): Figure 3.4 White mice embryonic stem cells after culture for days (A) Growth cone (TEM x 12600); (B) Cross-sectional cytoplasmic branches (TEM x 12600); (C) Synaptic (red circle) (TEM x 24100) - Immunohistochemical staining with markers Vimentin and TH When staining immunohistochemistry with marker vimentin, we observed that most of the cells were positive for vimentin marker in the cultures (Figure 3.5A), the cell nucleus was stained with Sytox blue (Figure 3.5B) The cells were less positive for the TH marker to mark dopamine-secreting neurons These dopamine-secreting neurons can stand alone or in clusters (Figure 3.5C, D) 15 Figure 3.5 HMMD staining with markers Vimentin and TH in white mice embryos after culture (fluorescence microscopy x200) (A, B) are different color phases in the same position; A: Vimentin marker positive cells (red); B: Cytox-positive cell nucleus (green) (C, D) TH marker positive cells c Percentage of cells, cell clusters positive for TH marker after culture Counting neurons and neuron clusters that were positive for TH marker by fluorescence microscopy, we found that the proportion of neurons and neuron clusters with the presence of Tyrosine Hydroxylase enzyme was as follows: Table 3.3 Percentage of cells, cell clusters positive for TH marker after culture: Percentage of TH Percentage of TH positive Embryo positive cell (+) cell cluster (+) age X ± SD (%) X ± SD (%) E10,5 1,18 ± 0,48 13,27 ± 7,40 E11,5 E12,5 5,38 ± 3,56 37,09 ± 6,53 E13,5 0,01 0.05 The survival rate after cryopreservation of group C was significantly lower with the other groups with p 0.05 The sample growth rate in group C was lower than with the other groups with p 0,05 19 7,5 week 1,08 ± 0,20 x 105 85,6 ± 5,0 0 week 0 11,5 week 102 Total Among 102 embryos isolated from neural tube epithelial cells, on average, embryo at 6,5 weeks old had a cell count of about 0,96 x 10^5 cells; 7-week embryo about 1,02 x 10^5 cells; 7,5 weeks old embryo is about 1,08 x 10^5 cells The survival rate of cells after isolation ranged from 84,7% - 86,1% There were no differences in the number of cells obtained and the cell survival in the isolated embryos 3.2.3 Proliferative and differentiation of human embryonic neural tube epithelial stem cells that produce dopamine-secreting neurons - Number of cultured cells positive for the TRY marker The number of TH-positive neurons in the cultures was assessed by embryo age and by culture time The results are shown in Table 3.7: 19 Table 3.7 Distribution of TH (+) cells in culture wells according to embryo age: days after days after 10 days after cultured cultured cultured Embryo Number Sample Sample Sample Sample Sample age of size (n) size size size size TH(+)cell 6,5 15,9 ± 40,7 ± 107,6 ± 18 18 18 week 4,8 17,9 10,04 18,4 ± 44,7 114,7 ± 65 65 65 week 5,7 ±15,9 16,4 7,5 17,4 ± 38,2 ± 111,8 ± 19 19 19 week 5,6 15,5 14,4 P=0,243 P=0,266 P=0,096 102 102 102 Total > 0,05 > 0,05 > 0,05 In culture time: the number of TH-positive cells increased from days, days and the largest was 10 days after cultured In embryo age: the number of TH-positive cells did not differ between cultures at different embryo ages from 6,5 to 7,5 weeks The increase in the number of TH marker-positive cells during culture is more clearly shown on the graph below: Chart 3.3 The increase in the number of TH(+) cells over time TH marker-positive cells tend to increase gradually after 5; and 10 days of culture The number of TH (+) cells increased from an average of about 17,8 ± 5,6 cells on day to 42,8 ± 16,3 TH-positive cells on day And at about day 10 after culture with an average of 112,9 ± 15,3 TH(+) cells 20 Chapter DISCUSSION 4.1 Isolation, proliferation, differentiation and cryopreservation of mice embryonic neural ectoderm stem cells 4.1.1 Isolation, proliferation culture of midbrain ectodermal stem cells according to embryo age group The time for the appearance of dopamine-secreting stem cells in the midbrain of mice embryos is about 10,5 days Therefore, to use this source of stem cells to create dopamine-secreting neurons at the embryonic age with the highest efficiency is our first question This study shows that (1) in terms of extraction, at the small embryo stage E10.5 the rate of dissection of midbrain samples is the lowest (72%) while at E12.5 – 14,5 this rate up to 90% (table 3.1); (2) in terms of total number of cells after isolation: in the E10.5 and E11.5 embryo age groups, the number of cells obtained was significantly lower than in the E12,5 embryonic age group; (3) in terms of culturability, only 10.5% of cell samples of E10,5 stage embryos were able to proliferate in culture medium while this percentage was 100% at E12,5 embryo age It can be seen that at a young age, the neural tube wall is thin, the dissection of stem cells is more difficult in the large embryonic age group, fewer cells are obtained and the proliferation culture efficiency is less Therefore, we have selected embryo age E12,5 – E13,5 to culture neural ectoderm stem cells 4.1.2 Proliferation and differentiation of mice embryonic neural ectoderm stem cells E12,5 - E13,5 generates dopamine-secreting neurons a Evolution of cultures over time The average time for cells to grow to the bottom of the culture cage in the study was 7,4 ± 2,3 days The mean timeline is quite similar to many studies According to the author Shimoda et al (1992), the midbrain floor stem cells develop best in the period of 5-10 days, after 14 days, the degeneration of cultured cells begins to be observed b Identification of cells after culture Golgi II staining to detect cells of neural tissue: many cell types were observed in the culture plate, mostly multipolar neurons with many long cytoplasmic branches and astrocytes Under scanning electron microscopy, images of bipolar neurons with oval cell bodies in the center or multipolar neurons with large cell bodies and many cytoplasmic branches are observed In addition, neuroblast-like cells and highly differentiated cells with synapses appeared in cell cultures This feature was also described by the author group Robert F et al (2012) when 21 culturing mice embryonic neural stem cells The authors also began to observe a small number of synapses after days of culture and a large increase after 14-21 days 4.1.3 Cryopreservation of embryonic neural ectoderm stem cells In this study, we used storage medium containing 10% DMSO - a cryoprotectant that penetrates the cell membrane This medium has been proven by many studies to have a good effect on neural stem cells such as: high cell survival rate after thawing, simple manipulation process, and no effect on the "original" nature of the neural stem cells cell Through the observational study, the best samples were kept refrigerated in the post-isolation and primary culture stage (P0), while the secondary culture stage (P1) gave a much lower survival rate So, is the cryopreservation capacity of mature neurons worse than that of immature cells? The authors' group Rodriguez et al (2017) also made a similar statement when the survival rate after thawing of the uncultured group was about 60-70% while in the group that had been cultured to create adult neurons (29-34%) Scientists agree that differentiated cells have a lower tolerance to hypothermia than undifferentiated cells, so cell membranes are more susceptible to damage leading to lysis cells during thawing The thawed cells were cultured to proliferate, resulting in the creation of a neuronal model with many stellate cells, oligodendrocytes and neurons This shows that cryopreservation does not affect cell proliferation and differentiation 4.2 Isolation, proliferation and differentiation of human embryonic neural ectoderm stem cells 4.2.1 Characteristics of embryos in the study The distribution of embryo age in the study was not uniform: 79,1% of embryos were at the stage of 6,5 – 7,5 weeks This is because this is the ideal time to perform the embryo reduction technique – the embryo is neither too small nor too large – which makes it easier to manipulate and does not affect the remaining embryos Moreover, in terms of time, this is the right time to take neural ectoderm stem cells to culture and differentiate to create dopamine-secreting neurons Authors Freeman et al (1991) when studying the reproduction and migration of dopaminesecreting neurons in human embryos from 6,0 to 13,2 weeks of age, found that dopamine-secreting receptor cells begin to appear in the midbrain ventral region from 5,5 weeks of age; around weeks of age cells begin to migrate out of the midbrain; weeks old observed the 22 formation of the neural network of dopamine-secreting neurons; and weeks old began to see the migration of dopamine-secreting neurons to the striatum and decreased expression of TH (+) neurons in the midbrain The use of mitogenic embryos for neural stem cell isolation has many advantages such as: knowing the exact age of the embryo, the embryos are obtained under aseptic conditions, the tissue sample is relatively pure, and finally, this is discarded biological products 4.2.2 Total number of isolated cells and survival The mean total number of cells isolated from an embryo ranged from 0,96 x 105 to 1,08 x 105 cells Among them, the survival rate after isolation of the samples reached over 80% Among the embryonic age groups of 6,5 weeks, weeks and 7,5 weeks, the embryo age group of 7,5 weeks had the largest number of cells, but this difference was not statistically significant 4.2.3 Proliferative culture and differentiation of embryonic neural tube epithelial stem cells that generate dopamine-secreting neurons After 2-3 days of culture, observed under the stereo microscope, the cells began to stick to the bottom of the culture cage, the cells tended to differentiate into neurons when many cytoplasmic branches radiated around Cells tended to have better basal adhesion in the 7,5-week embryos, possibly due to the higher number of cells isolated at baseline compared with the 6,5-week-old embryos The time for cells to cover the bottom of the culture cage is about 7-10 days Compared with mice embryonic cultures, human embryonic stem cells appear to have a slower proliferative rate, although the percentage of cells in initial culture is similar 4.3 Effect of culture on creating dopamine-secreting neurons In this study, the TH marker was used to mark dopamine-secreting neurons in post-culture cell samples In cultures of mice and human embryos, the presence of TH(+) cells was observed, which tended to increase over time The percentage of TH(+) cells in the mice embryo sample was 5,38 ± 3,56% This percentage varies in many studies, reported high ranging from to 18% The reason for such a difference is due to the heterogeneity in culture medium, culture method and embryo age used for culture In the study of the author McKay et al (1998), the addition of 10% FBS and bFGF to the culture medium will give a higher rate of recovery of dopamine-secreting neurons than in the no-addition group (14,1% with 2,9%) The author Stull et al (2002) brought up the 23 idea of researching the use of antioxidants in culture medium such as vitamin E, Selenite, melatonin for the purpose of protecting neurons against oxidative stress The results were not unexpected when the authors found that the group that supplemented these substances in the culture medium had the ability to grow for at least days longer than the group without supplementation as well as a number of dopaminesecreting neurons times higher than in the antioxidant supplement group CONCLUSION From the results obtained from the study of isolation, proliferation and differentiation of embryonic - fetal neural stem cells into dopaminesecreting cells, we draw the following conclusions: Isolation, proliferation, differentiation and cryopreservation of embryonic neural ectoderm stem cells in mice Isolation, proliferation and differentiation were best at embryonic age E12,5 –E13,5 The rate of isolation of middle brain tissue samples was 100% successful with the number of cells after isolation about 1,34 ± 0.048 x 10^5 cells Characteristics of post-cultured cells: as cells of nervous tissue, many cells have cytoplasmic branches radiating around and contact with adjacent cells Using the TH marker to mark dopamine-secreting neurons, many TH-positive cells were observed in the post-culture sample The percentage of neurons positive for TH is about 5,38 ± 3,56% Cryopreservation of embryonic neural stem cells in DMEM 10% DMSO gives a survival rate of about 64,9% Cells in post-isolation or primary culture showed higher survival rates after cryopreservation than cryopreservation in secondary culture Isolation, proliferation and differentiation of human embryonic neural stem cells: Neural tube embryos of human embryos obtained from fetal reduction techniques in assisted reproductive therapy can be used to culture and generate dopamine-secreting neurons On average, embryo at 6,5 weeks old had a cell count about 0,96 x 10^5 cells; 7-week embryo about 1,02 x 10^5 cells; 7,5 weeks old embryos is about 1,08 x 10^5 cells The survival rate of cells after isolation is 86,1 ± 3,7%; 84,7 ± 4% and 85,6 ± 5% for each state 24 Characteristics of post-cultured cells: as cells of nervous tissue, many cells have cytoplasmic branches radiating around and contact with adjacent cells Using the TH marker to mark dopamine-secreting neurons also observed many TH-positive cells in the post-culture sample The number of TH-possitive cells after and 10 days of culture increased from 15,9 ± 4,8 cells to 107,6 ± 10,04 cells at 6,5 weeks; from 18,4 ± 5,7 cells to 114,7 ± 16,4 cells at weeks and from 17,4 ± 5,6 cells to 111,8 ± 14,4 cells at 7,5 weeks RECOMMENDATIONS AND DIRECTIONS FOR FURTHER RESEARCH In terms of research on human embryos, we found that there are still some difficulties in embryo collection as well as in isolating embryonic neural ectoderm stem cells due to the incomplete characteristics of the post-mitotic embryos Therefore, more research is needed on human embryos to perfect the culture process and increase the rate of dopamine-secreting neurons The goal of the study is to culture to create dopamine-secreting neurons for the treatment of Parkinson's disease Therefore, further studies are needed on treatment trials in animals as well as in humans to evaluate the viability of cells after culture in the host brain ... post-transplant mice However, this technique revealed many limitations such as: low survival rate of mice after transplantation, symptom improvement rate in heterogeneous study batches These limitations... the method of pregnancy reduction in women with multiple pregnancies during Assisted Reproductive Technology treatment and with the consent of the pregnant woman 2.1.2 Place and time of study... attenuated embryos from pregnant women undergoing IVF and artificial insemination with multiple pregnancies (≥ pregnancies) The distribution of embryo age and the ability to dissect and isolate the embryos