A METHOD AND CULTURE MEDIUM FORMULATION THAT CAN BE USED IN OBTAINING MOTOR NEURONS FROM NEUROMESODERMAL STEM CELLS DESCRIBED FROM PLURIPOTENTE STEM CELLS.

Abstract

This invention relates to a culture medium and a method for obtaining motor neuron cells from human pluripotent stem cell-derived neuromesodermal stem cells (NMCs). Furthermore, the present invention relates to the production of motor neuron cells using this culture medium and method, which can be used in research and clinical applications.

Description

25418.127 1 TARIFF PLUROPOTENT STEM CELL-DERIVED NEUROMESODERMAL PRODUCTION OF MOTOR NEURONS FROM STEM CELLS A METHOD AND CULTURAL ENVIRONMENT THAT CAN BE USED FORMULATION 5 Technical Area This discovery is made from neuromesodermal stem cells derived from human pluripotent stem cells. (NMK) is a culture medium for obtaining motor neuron cells. (culture medium) and relate to a method in which this medium is used. Furthermore, the present invention is 10 with this culture environment and this method in research and clinical applications It is related to obtaining usable motor neuron cells. Previous Technique The spinal nerves, located within the spine, are a 15% component of the central nervous system. It is part of the brain and body. By providing nerve transmission between the brain and body, it enables muscle movements and sensations. It enables the performance of autonomous functions such as transport and digestion. Spinal cord injury occurs as a result of mechanical damage. Damaged Inflammation and cell death in the region cause serious neuronal damage [1]. Spinal cord neuron damage results in irreversible functional impairments in 20 years. This results in a very low percentage, just 2.1%, of spinal cord injury cases. functional regeneration can be observed [2]. Spinal cord neurons are part of the central nervous system, and these neurons... Its capacity for self-renewal is quite low. This situation limits treatment options to 25 It restricts the elongation of the axons of neuron cells located in the damaged area. It contributes to tissue renewal, but allows for limited regeneration. Although it provides functional tissue regeneration, it is low [3]. In this context, nerve in the region In order to enable transmission, new neurons or neuron cells are needed. 25418.127 2 Transplanting cells that can differentiate to the damaged area is a cellular process. It is a treatment method. In studies previously reported in the literature, transplantation Although only a small fraction of the cells produced can differentiate into nerve cells, it is not sufficient. It is not functional and is not seen as functional [4]. For this reason, functional neuron Obtaining, maintaining in culture, and ensuring nerve transmission are extremely important. 5 The source from which cells will be obtained in culture and the acquisition of functional cells. This is extremely important in this context. Pluripotent stem cells; embryonic stem cells and induced (stimulated) stem cells. They are classified as pluripotent stem cells and, thanks to their pluripotent properties, 10 It can differentiate into many cell types. As a stem cell therapy product. to use pluripotent human stem cells that can differentiate into all cell types Various attempts have been made. In the literature, pluripotent stem cells... spinal cord neuron generation and neuromesodermal stem cells as an intermediate step Below are the patents relating to its potential use. The previous 15 Taking into account the information available in the field of technology, the culture described in the invention presented herein... We would like to inform you that there is no existing application similar to this environment and the developed protocol. The patents mentioned below relate to the production of functional neurons. They are different inventions. Patents under the prior art US7531354B2 WO2009122413A1, WO2014 / 148646, US20170283774A1, US20160068806A1 They are listed as follows. Brief Description of the Invention 25 This invention develops neuromesodermal stem cell (NMC) intermediate from pluripotent stem cells. a culture medium for obtaining functional neurons using the step It relates to the culture medium and a method used in employing this medium. 25418.127 3 This discovery is made from neuromesodermal stem cells derived from human pluripotent stem cells. a culture medium for obtaining motor neuron cells and this medium It relates to a method used. The present invention overcomes all the problems mentioned above and addresses the related 5 bringing additional advantages to the technical field and, under normal conditions, from adult individuals Unlimited production of motor neurons from pluripotent stem cells, which is otherwise impossible to obtain. a culture medium that allows its production and a medium in which this medium is used It is related to the method. The invention in question describes a method for the developmental progression of motor neurons in culture. functionally derived from neuromesodermal stem cells, which are a biological step. This is achieved by obtaining motor neurons from pluripotent stem cells. It allows for repeated generation. The current invention is a culture in which motor neurons can be passaged for extended periods of time. It relates to the environment and the method by which that environment is used. The developed method and culture medium formulation content were obtained for research purposes. embryonic stem cells obtained through therapeutic cloning or 20 induced pluripotent stem cells obtained through reprogramming For motor neuron differentiation via neuromesodermal stem cell intermediate step It is applicable. The developed method and culture medium formulation content was obtained from 25 adult individuals. Reprogramming and culturing of the obtained cells, neuromesodermal stem cells. It can be used for cell and motor neuron differentiation. 25418.127 4 The developed method and culture medium formulation content differentiates. It can be made into a kit for triggering and applied over time. It is designed as a kit, formulation, protocol or cell source. applicable. The developed method and culture medium formulation content are intended for cellular therapy purposes. It can be adapted to GMP standards and used in a customized way. The developed method and culture medium formulation content are used in motor neuron therapy. Nerve damage disease treatments, treatment of neurodegenerative diseases and general 10 It can be used in the treatment of nerve damage. An invention is a process within a cultural environment at specific time intervals. It is related to its usage. The invention in question involves a method that involves a culture at specific time intervals. Motor neuron cells are obtained through the medium, and they multiply and... Its characteristics are preserved. The present invention also describes human pluripotent stem cell-derived neuromesodermal stem 20 a culture that can be used for the differentiation of motor neuron cells from cells It is related to the formulation of the medium. This invention is relevant to all those involved in research and development (R&D, product development) processes. in centers (universities, research institutes, private sector companies conducting research 25 (based structures), products for cellular therapy research and application. in companies that prepare and commercially market products, Clinical Practice (GMP) in centers that carry out the relevant process (university hospitals, universities, GMP) centers), all centers, hospitals, stem cells that conduct processes with health practices 25418.127 If they use cell-derived materials, they indirectly benefit from the above-mentioned in collaboration with centers and also for neurological conditions such as spinal cord injury and neuronal damage Cellular therapy provides services for the treatment and rehabilitation of systemic diseases. It can be used in clinics and hospitals that can implement this. Brief Description of the Figures The purpose of this discovery was achieved through "Pluripotent Stem Cells". Obtaining Motor Neurons from Neuromesodermal Stem Cells A Usable Method and Culture Medium Formulation” in the attached figures. shown; of these figures: 10 Figure 1. Generating NMK from induced pluripotent stem cells and Characterization of NMK cells using bFGF and CHIR. It is done daily in a suspended culture medium. After three days, the result is obtained. The cells were tested for the NMK markers T / Bra and Sox-2 proteins. It shows positive staining. As a result, the obtained cells are 15 It is a NMK cell. Notes: bFGF (basic fibroblast growth factor), CHIR (CHIR99021-Wnt pathway activator), T / Bra (Brachyury), Sox-2 (SRY (sex determining region Y)-box 2). Figure 2. Obtaining spinal cord neurons from induced pluripotent stem cells. protocol steps and resulting neurons. The protocol in question is 20 Specific factors are added to N2B27 culture medium as indicated in the diagram. It was applied using [method]. Active neurons after fifteen days. obtained and showed activity. Embryoid in Figure B. Neurons extending outwards from the globules and forming over time is being observed. As a result, the implemented protocol is actively being used for 25 years. It has been studied and neurons have been obtained. Notes: bFGF (basic fibroblast) growth factor), CHIR (CHIR99021-Wnt pathway activator), RA (Retinoic acid), SAG (SAG dihydrochloride- Smoothened Agonist), Shh (Sonic Hedgehog), D: day 25418.127 6 Figure 3. Immunostaining of neuronal markers in the obtained neurons. and density measurements. Neurons obtained after fifteen days. are active, and these neurons have active neuron markers at the protein level. The staining was demonstrated. The staining results showed the presence of neurons. observed β-Tubulin, Isl-1, Isl-2, MNX-1, Neun, NF, Map2 5 The presence of proteins has been demonstrated. The neurons obtained as a result... It has motor neuron characteristics. Figure 4. Neurons rapidly adhering to the surface after thawing. In culture. To show that the obtained neurons can be frozen and stored. For this purpose, a freeze-thaw cycle was performed. Neurons 10 Once dissolved, it quickly adhered to the surface and exhibited a morphological characteristic. No changes occurred. The motor neurons obtained as a result. In culture, it can be stored by freezing. Figure 5. Axon elongation of the obtained spinal nerves. Protocol application. The neurons obtained as a result were transplanted from the same region for a period of 3 days. axon elongation and the dynamic structure of neurons when monitored It is observed that neurons are elongating and forming connections. Result as neurons undergo axonal elongation and synapse (between neurons) It has been observed that it forms a connection. Notes: D (day) Figure 6. Synaptic vesicles and neuronal transmission. The resulting neurons interact with each other 20 it establishes connections in a network-like manner, and synaptic vesicles are formed at these connections. It has been observed that this occurs. This situation involves active neuron formation and nerve development. It is an indicator of transmission. The arrows show synaptic vesicles. Detailed Description of the Invention 25 The present invention is a neuromesodermal stem cell derived from human pluripotent stem cells. a culture medium for obtaining motor neuron cells from cells and This relates to a method used in this environment. 25418.127 7 The present invention allows motor neuron cells produced by this method to be used in spinal cord injury or Cellular therapy products that can be used in neuron damage applications and in human For the production of motor neuron cells from pluripotent stem cells standardized and optimized for use in clinical applications. It involves an introduced method of acculturation and a cultural environment. 5 As used here, the term "stem cells" refers to the cells of tissues and organs. It means master cells that can regenerate indefinitely in order to create Stem cells can be totipotent, multipotent, pluripotent, oligopotent, or... They are unipotent cells. 10 As used here, the term "pluripotent stem cells" refers to cells that can be extracted from a living body. pluripotent stem cells that can differentiate into all three germ layers that make up the stem This corresponds to and examples of these are embryonic stem cells and It contains induced pluripotent stem (iPS; UPK) cells. The 15 in question... the method includes induced pluripotent stem cells and embryonic stem cells. It can be adapted and used with all pluripotent stem cells, including those mentioned above. In the preferred application of the invention, via neuromesodermal stem cells Human induced pluripotent stem cells for the generation of motor neurons It has been used. 20 As used here, the term "differentiation" refers to both embryonic development and During cell division, multiplication, and growth in an adult individual, the cells It refers to a process that specializes in structure or function. As used here, the term "embryoid globule" refers to a pluripotent stem. It refers to an aggregate formed by induced differentiation of cells. 3D They are structures. 25418.127 8 Neuromesodermal stem cell therapy to treat spinal cord injuries at the cellular level. The cells can differentiate into neuron (nerve) cells in the spinal cord. The invention in question involves neuromesodermal stem cells derived from human pluripotent stem cells. a culture medium for the differentiation of NMC cells into motor neuron cells formulation and use of this culture medium in the specified method 5 It includes. In the present invention, the culture medium formulation is in N2B27 serum-free agar. DMEMF12 / neurobasal medium (1:1), Bovine Serum Albumin (BSA), N2 supplementation, B27 addition, penicillin (10,000 U / ml), streptomycin (10,000 μg / ml) and L-glutamine 10 It contains the following substances; in addition, bFGF (essential fibroblast growth factor; It contains basic fibroblast growth factor (Wnt activator) and CHIR-99021 (Wnt activator). In the preferred application of the invention, the concentration of bFGF in the culture medium The concentration of CHIR-99021 is 12 ng / ml and 3 µM. The prescribed doses are fixed and 15 It should be used in this way. The invention in question involves induced pluripotent stem cells in serum-free culture medium. The cells were treated with 12 ng / ml bFGF and 3 µM CHIR-99021 for three days. These 20 cells were differentiated into neuromesodermal stem cells (NMCs) (Figure 1). Differentiation was performed in suspension culture medium and embryoid spherules were obtained. It has been done. The NMK phenotype of the differentiated cells was determined by T / Bra and Sox-2 staining. This has been demonstrated and proven (Figure 1). 25 In the preferred application of the invention, the culture medium formulation is N2B27. DMEMF12:neurobasal medium (1:1), 0.5% Bovine in serum-free culture medium 25418.127 9 Serum Albumin (BSA), 0.5% N2 supplementation, 1% B27 supplementation, 1% penicillin (10,000 It contains (U / ml)-streptomycin (10,000 μg / ml) and 1% L-glutamine. Neuromesodermal stem cells cultured in N2B27 (serum-free medium) In the environment shown in Figure 2A, 5 neurons in the spinal cord were connected. It has been differentiated. The time-dependent steps used in the method and the applied methods The culture medium content is shown in Table 1. Table 1. Method steps and content. Time Differentiation Method Day 0 - Day 3 12 ng / ml bFGF + 3uM CHIR Day 3 – Day 5 0.1 uM RA + 0.5 uM SAG Day 5 – Day 7 0.1 uM RA + 50 ng / ml Shh Day 7 – Day 8 0.1 uM RA + 50 ng / ml Shh+10 ng / ml bFGF Day 8 and onwards: 10 ng / ml bFGF The obtained neurons had an active neuron phenotype by day 15. (Figure 2C). These neurons were passaged in culture by scraping and kept for up to 40 days. This has made it possible to maintain the neurons in culture. Cells that form embryoid globules in suspension culture until the third day On the third day, it is transferred to a surface coated with a matrigel-gelatin mixture and adheres for 15 days. This was achieved, and culture was subsequently realized in a two-dimensional culture. The term "cultural environment" as expressed here refers to bFGF, CHIR, RA, SAG and It includes cultural environments that do not contain Shh. 25418.127 The neurons obtained were labeled β-Tubulin, Isl-1, Isl-2, Minx1, NeunN, NF, Map-2. Neuronal characteristics were characterized by immunostaining with markers. Figure This shows staining with 3 neuron markers. The resulting neurons were cultured in N2B27 5, which contains bFGF and is also the culture medium. freeze-thaw with a solution obtained by adding 10% DMSO to the culture medium. and has been able to maintain its viability (Figure 4). The freezing process involves scraping the cells and by precipitating and suspending in freezing solution This was carried out. The prepared vials were kept overnight in a tank with isopropanol at -80 degrees. After being left to stand, it was stored in a nitrogen tank at -196 degrees. 10 The resulting neurons have a dynamic structure in culture and undergo axonal elongation. This is happening. This is the first indication that these neurons are showing functional activity. is an indicator (Figure 5). The obtained neurons showed synaptic vesicles indicating nerve transmission between axons, and Transmission in axons has been observed. This indicates a functional neuron activity. is an indicator (Figure 6). The present invention involves the development of neuromesodermal stem cells from human pluripotent stem cells. A culture medium for differentiation into motor neurons via (NMK) This is a method for culturing stem cells using the following steps: It includes: a) Suspended serum-free N2B27 of induced human pluripotent stem cells 25 treated with bFGF and CHIR-99021 in culture medium Differentiation into neuromesodermal stem cells (NMC) b) The resulting neuromesodermal stem cells are cultured on a surface in the culture medium. maintaining 25418.127 11 c) functional motor neurons of implanted neuromesodermal stem cells Serum-free N2B27 to enable differentiation (acquisition) of cells RA and SAG; RA, Shh and beGF; beGF added to the cultural environment - respectively. It is done. In step (a) of the method described in the invention, induced human pluripotent stem cells 12 Serum-free N2B27 culture prepared by adding ng / ml bFGF and 3 µM CHIR-99021. in medium and in suspension culture for 3 days at 37°C temperature and 5% CO2 It is cultured in an incubator with a specific atmosphere. (Figure 1) 10 Neuromesodermal roots formed in step (a) in step (b) of the invention method The cells are attached to a surface. This step, Transfer of neuromesodermal stem cells from 3D culture to 2D culture It enables transfer. 15 obtained in suspended culture up to the third day. The harvested cells were coated with a matrigel-gelatin mixture on the third day. It was transmitted and its hold was ensured, and culture subsequently existed in a two-dimensional culture. This was achieved by placing the cells on the surface after 3 days of suspension culture. It undergoes differentiation by adhering to each other (Figure 1). In step (c) of the method that is the subject of the invention, after the retention process in step (b) 0.1 µM RA + 0.5 µM SAG were added to the N2B27 culture medium between days 3 and 5; 0.1 µM RA + 50 ng / ml Shh between days 7 and 8, and 0.1 µM RA + 50 ng / ml Shh between days 7 and 8. Continue the culturing process by adding uM RA + 50 ng / ml Shh + 10 ng / ml bFGF. It is done. After incubation until day 8, 25 samples with added 10 ng / ml bFGF. The cultural environment is provided. In this step (c), cell differentiation continues until day 15, and the following is obtained The neurons that were isolated had developed an active neuron phenotype by the 15th day. 25418.127 12 The aim is to induce a transition of cells to the functional motor neuron cell phenotype. The protocol steps have transitions like this. These neurons are scraped in culture. By passage through the method, it can be brought from day 15 to day 40, and the method Active neurons are obtained on day 15, and this process can be extended for a total of up to 40 days. This is the protocol (Figure 1). 5 Motor neuron cells produced using the method described in the present invention can be used for spinal cord injuries or It possesses characteristics that could be used in cell therapy for neuronal damage. The following examples are intended to better illustrate the concept of invention, and the subject of invention is these 10 This is not limited to examples. EXAMPLES Example 1-15 Neuromesodermal cell production technique: Within the scope of this invention, neurons Neuromesodermal stem cells were used as a preliminary step to obtain them. These cells are technically pluripotent stem cells suspended in culture for three days. obtained by incubating in a medium containing bFGF and CHIR without any retention throughout the process. NMK cells are thus obtained as embryoid spherules (Figure 1). (Figure 2). The resulting in vivo counterparts, NMK cells, were T / Bra cells. and Sox2 factors are described simultaneously (Figure 1). Generating neurons from NMK cells: NMK cells are suspended. Although obtained in the medium, neuronal differentiation of these cells is done in a single layer 25 It was produced in a cell culture medium. Coated with a Matrigel-gelatin mixture. Embryoid microspheres transferred to surfaces adhere to the surfaces, as shown in Figure 2. They were exposed to a differentiation protocol and neurons were formed. 25418.127 13 The resulting neurons become active neurons by elongating their axons during the differentiation program. It shows the morphology (Figure 2 and Figure 5) and lipid vesicle movements. nöron iletimi yapmaktadır (Şekil 6). 25418.127 14 REFERANSLAR [1]. Zhang, N., Yin, Y., Xu, S. J., Wu, Y. P., Chen, W. S. 2012. “Inflammation & apoptosis in spinal cord injury”, The Indian Journal of Medical Research, 135(3), 287-296. 5 [2]. Shroff, G. 2016. “Human embryonic stem cell therapy in chronic spinal cord injury: a retrospective study”, Clinical and Translational Science, 9(3), 168-175. [3]. Dell’Anno, M.T., Strittmatter, S. M. 2016. “Rewiring the spinal cord: direct and indirect strategies”, Neuroscience Letters, 652, 25–34. [4]. Abematsu, M., Smith, I., Nakashima, K. 2006. “Mechanisms of neural stem cell fate determination: extracellular cues and intracellular 15 programs”, Current Stem Cell Research & Therapy, 1, 267–277.

Claims

25418.127 REQUESTS 1. Human pluripotent stem cells and neuromesodermal stem cells (NMC) a culture medium for differentiation into motor neurons through It is a method for culturing stem cells using the following 5 methods: It includes the following steps: a) Suspension of induced human pluripotent stem cells in serum-free N2B27 treated with bFGF and CHIR-99021 in culture medium Differentiation into neuromesodermal stem cells (NMC) b) The resulting neuromesodermal stem cells are cultured on a surface in the culture medium at 10°C. maintaining c) Functional motor of implanted neuromesodermal stem cells serum-free to enable differentiation (acquisition) into neuron cells The following substances were added to the N2B27 culture medium: RA and SAG; RA, Shh and beGF, respectively; The addition of beEGF. 15 2. It is a method according to claim 1, and its characteristic is that a) step 3 is up to day 3, b) on day 3 of step c) from day 3 of step 8 to day 40 It is the continuation of.

3. A method according to Claim 2, characterized by the fact that the method takes a total of 15 to 40 days. It is the continuation.

4. It is a method according to claim 3, and its characteristic is that in step (a), bFGF concentration of 12 ng / ml and CHIR-990213 concentration of 3 µM 25 It is the fact that.

5. It is a method according to claim 4, and its characteristic is the culture involved in step (a). DMEMF12:neurobasal medium (1:1), 0.5% Bovine Serum Albumin 25418.127 16 (BSA), 0.5% N2 supplement, 1% B27 supplement, 1% penicillin (10,000 U / ml) Containing streptomycin (10,000 μg / ml) and 1% L-glutamine. The culture medium should be serum-free N2B27.

6. It is a method according to claim 5, and its characteristic is that in step c), the culture medium is sequentially 5. and the additions are made as follows: - Between days 3 and 5, add 0.1 µM RA and 0.5 µM SAG. - Between days 5 and 7, add 0.1 µM RA + 50 ng / ml Shh. - Between days 7 and 8, 0.1 µM RA + 50 ng / ml Shh + 10 ng / ml Addition of bFGF 10 - The addition of 10 ng / ml bFGF between days 8 and 40.

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