KR20210051508A - Screening Method of Therapeutic Stem Cells for the Treatment of Neurological Disorders Using 3-Dimensional Brain Organoid - Google Patents

Abstract

The present invention relates to a method for screening stem cells effective for the treatment of neurological disorders, and more specifically, to an evaluation platform that mimics the state of neurological disorders using a three-dimensional brain organoid, and treats the same with stem cells to compare and analyze the therapeutic effects, thereby predicting the therapeutic effects of the stem cells in advance and selecting only stem cells effective for treatment. By providing the method for screening stem cells according to the present invention, compared to existing methods in which stem cell treatment was applied blindly without verification, the treatment effects are innovatively improved and the uniformity of the treatment result is increased. Ultimately, it is expected to suggest an alternative that the stem cell treatment can be used universally.

Description

[Screening Method of Therapeutic Stem Cells for the Treatment of Neurological Disorders Using 3-Dimensional Brain Organoid}

The present invention relates to a method for screening stem cells for the treatment of cranial nervous system diseases, and more specifically, to a method of screening stem cells effective for treating cranial nerve diseases by using a three-dimensional brain organoid.

Among the stem cell technologies being developed recently, when not only differentiates stem cells into specific cells, but also induces differentiation into a 3D state in the process of differentiating into a specific cell, an organoid in a 3D state that has a structure similar to that of body organs. (Organoid; pseudo-organ) structure, and based on these findings, to date, brain organoid, heart organoid, liver organoid, lung organoid (lung organoid), small intestine organoid (small intestine organoid), and various mini-organoids can be produced has been reported. The development of these pseudo-organs, that is, organoids, shows the creation of a 3D environment close to the in vivo, so that drugs can be tested as if they were acting in the'inside' even in the'outside' experimental situation that could not be seen before. Rather, it has the advantage of being able to reproduce the effects that appear in real human organs as it is.

On the other hand, in the treatment of cranial nervous system diseases such as Alzheimer's disease, Parkinson's disease, cerebral infarction, cerebral hemorrhage, and spinal cord injury, a variety of new therapeutic candidates have appeared through the regeneration of nerve cells, and such solutions include embryonic stem cells and pluripotency. Various methods for treating cranial nervous system diseases have emerged by creating and administering cells for patient-specific treatment using stem cells or the like. However, the main reason stem cell therapy has not yet become common is that there are cases in which the treatment results are different because the treatment efficacy is not the same for each donor stem cell. However, no evaluation system has been developed to select stem cells that can predict treatment efficacy, and research on this part is relatively insufficient.

Hee Jin Kim et al., Stereotactic brain injection of human umbilical cord blood mesenchymal stem cells in patients with Alzheimer's disease dementia: A phase 1 clinical trial, Alzheimers Dement (N Y). 2015 Sep; 1(2): 95-102.

The present invention has been conceived to solve the above-described problems, and an object of the present invention is to provide a stem cell screening method for treating cranial and nervous system diseases, comprising the following steps:

(a) preparing a 3-Dimensional brain organoid using human dedifferentiated stem cells;

(b) treating and culturing the brain organoids with amyloid beta oligomers and candidate stem cells at the same time; And

(c) selecting a stem cell having a therapeutic effect from among the candidate stem cells.

Another object of the present invention is to provide a method for screening stem cells for treatment of cranial and nervous system diseases, comprising the following steps:

(a) preparing a three-dimensional brain organoid using human dedifferentiated stem cells;

(b) culturing the brain organoids;

(c) treating and culturing the brain organoids with amyloid beta oligomers and candidate stem cells at the same time; And

(d) selecting a stem cell having a therapeutic effect from among the candidate stem cells.

Another object of the present invention is to provide a method for screening stem cells for treatment of cranial and nervous system diseases, comprising the following steps:

(a) preparing a three-dimensional brain organoid using human dedifferentiated stem cells;

(b) culturing the brain organoids;

(c) treating and culturing the brain organoids with amyloid beta oligomers and candidate stem cells at the same time;

(d) selecting stem cells having a therapeutic effect from among the candidate stem cells; And

(e) comparing the treatment effect by treating the selected stem cells to a cranial nerve disease model animal.

However, the technical task to be achieved by the present invention is not limited to the above-mentioned tasks, and other tasks that are not mentioned can be clearly understood by those of ordinary skill in the technical field to which the present invention belongs from the following description. will be.

In order to achieve the object of the present invention as described above, the present invention provides a stem cell screening method for the treatment of neurological disorders, including the following steps:

(a) preparing a 3-Dimensional brain organoid using human dedifferentiated stem cells;

(b) treating and culturing the brain organoids with amyloid beta oligomers and candidate stem cells at the same time; And

(c) selecting a stem cell having a therapeutic effect from among the candidate stem cells.

In one embodiment of the present invention, the three-dimensional brain organoid of step (a) may be prepared using 9×10 ³ to 1×10 ⁴ human dedifferentiated stem cells.

In another embodiment of the present invention, the amyloid beta oligomer of step (b) may be treated at a concentration of 8 to 12 μM for 2 to 4 days, preferably at a concentration of 10 μM for 3 days. .

In another embodiment of the present invention, the candidate stem cells of the step (b) may be co-cultured with the brain organoids ^{of 1 × 10 4} to 5 × 10 ^{4 cells.}

In another embodiment of the present invention, the candidate stem cells in step (b) may be derived from different donors or may be derived from different types of tissues of the same donor.

In another embodiment of the present invention, in the step (c), the group in which the death of neurons occurs the least, the group in which the expression of tubulin-beta III is the least decreased, or the nestin It may be to select the stem cells of the group with the least decreased expression.

In another embodiment of the present invention, the cranial nervous system disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, Batten disease, It may be one or more selected from the group consisting of cerebral infarction, cerebral hemorrhage, and spinal cord injury.

The present invention also provides a stem cell screening method for the treatment of cranial and nervous system diseases, comprising the following steps:

(a) preparing a three-dimensional brain organoid using human dedifferentiated stem cells;

(b) culturing the brain organoids;

(c) treating and culturing the brain organoids with amyloid beta oligomers and candidate stem cells at the same time; And

(d) selecting a stem cell having a therapeutic effect from among the candidate stem cells.

In one embodiment of the present invention, the culture of step (b) may be cultured in a medium containing mTeSR1, N2 supplement, and MEM NEAA (Non-Essential Amino Acid), preferably mTeSR1, N2 It can be cultured in a medium containing supplements, MEM NEAA and heparin.

In another embodiment of the present invention, the cultivation of step (b) may be performed for 30 to 50 days, and preferably may be performed for 30 to 40 days.

In addition, the present invention provides a stem cell screening method for the treatment of cranial and nervous system diseases, comprising the following steps:

(a) preparing a three-dimensional brain organoid using human dedifferentiated stem cells;

(b) culturing the brain organoids;

(c) treating and culturing the brain organoids with amyloid beta oligomers and candidate stem cells at the same time;

(d) selecting stem cells having a therapeutic effect from among the candidate stem cells; And

(e) comparing the treatment effect by treating the selected stem cells to a cranial nerve disease model animal.

In one embodiment of the present invention, the stem cell screening method according to the present invention comprises the step of finally selecting a stem cell having a higher therapeutic effect compared to other candidate stem cells in both steps (d) and (e). It may contain more.

In the case of using the stem cell screening method according to the present invention, stem cells that are substantially effective in the treatment of neurological diseases in vitro can be effectively selected, thereby maximizing the effect of stem cell treatment for patients with neurological disorders. In addition, compared to the method of applying stem cell treatment with blind without existing verification, it innovatively improves the treatment effect and improves the uniformity of treatment results, ultimately suggesting an alternative that stem cell treatment can be used universally. It is expected to do.

1 is a diagram showing the result of co-treatment of amyloid beta oligomer (Aβ) alone or with amyloid beta oligomer and human inferior turbinate-derived mesenchymal stem cells (hNTSCs) in a three-dimensional brain organoid.
2 is a view confirming the expression of tubulin-beta III, TUNEL, and Iba-1 according to stem cell treatment in an in vitro Alzheimer's disease model using a three-dimensional brain organoid by immunofluorescence staining.
3 is a view confirming the histological analysis of neurons according to the treatment of stem cells derived from different tissues or stem cells from different donors in an in vitro Alzheimer's disease model using 3D brain organoids by H&E staining.
4A and 4B are diagrams confirming the expression of Nestin (FIG. 4A) and Tuj1 (FIG. 4B) in neurons according to various types of stem cell treatment in an in vitro Alzheimer's disease model using 3D brain organoids. to be.
FIG. 5 is a diagram showing changes in expression of proteins by performing cytokine analysis after processing various types of stem cells in an in vitro Alzheimer's disease model using 3D brain organoids.
6A to 6C are diagrams showing the results of verifying the stem cell screening method according to the present invention using an Alzheimer's disease animal model, FIG. 6A shows the expression level of amyloid beta, and FIG. 6B shows the Morris water maze (Morris water maze). ) Test results, Figure 6c is a diagram showing the expression level of TIMP-2.

The present inventors established a reproducible Alzheimer's disease model in vitro using three-dimensional brain organoids, and selected stem cells effective for the treatment of Alzheimer's disease using this, and the selected stem cells are also in vivo Alzheimer's model animals. The present invention was completed by confirming that it exhibited the same therapeutic effect.

In one embodiment of the present invention, a three-dimensional brain organoid was produced from human dedifferentiated stem cells, and an in vitro Alzheimer's disease model was established using this (see Examples 1 and 2).

In another embodiment of the present invention, as a result of evaluating the treatment efficacy of stem cells using the three-dimensional brain organoid, it was confirmed that the treatment efficiency of mesenchymal stem cells derived from the inferior turbinate of the donor 376 was the best (Example 4 And 5).

In another embodiment of the present invention, it was confirmed that the expression difference of various proteins such as TIMP1, TIMP2, OPN, OPG, and IL-6 appeared according to the treatment of stem cells in an in vitro Alzheimer's disease model using a three-dimensional brain organoid. (See Example 6).

In another embodiment of the present invention, the treatment efficiency of mesenchymal stem cells derived from the inferior turbinate of the donor 376 was the best even in an in vivo Alzheimer's model animal experiment, consistent with the in vitro evaluation results using the three-dimensional brain organoids. Was confirmed (see Example 7).

Therefore, the stem cell screening method according to the present invention has established a reproducible standardized platform in selecting stem cells for the treatment of cranial and nervous system diseases, and can reduce the uncertainty of the treatment result using stem cells, and ultimately, a stem cell treatment agent. Is to be universally used in the treatment of various cranial and nervous system diseases.

Hereinafter, the present invention will be described in detail.

The present invention can provide a stem cell screening method for the treatment of neurological diseases, including the following steps.

(a) preparing a 3-Dimensional brain organoid using human dedifferentiated stem cells;

(b) treating and culturing the brain organoids with amyloid beta oligomers and candidate stem cells at the same time; And

(c) selecting a stem cell having a therapeutic effect from among the candidate stem cells.

In the present invention, the term "stem cell" refers to a cell that has the ability to differentiate into two or more different types of cells while having the ability to self-replicate as an undifferentiated cell.

In the present invention, “reversely differentiated stem cells” include induced pluripotent stem cells (iPS cells) generated by reprogramming somatic cells by expression or induction of expression of a reprogramming factor. Dedifferentiated stem cells can be produced using fetal, postnatal, neonatal, juvenile or adult somatic cells. Dedifferentiated stem cells can be newly generated (by methods known in the art) before differentiation into RPE cells or other cell types begins. Dedifferentiated stem cells can be specifically generated using material from a specific patient or matched donor for the purpose of generating tissue-matched RPE cells. Dedifferentiated stem cells can be prepared from cells that are not substantially immunogenic in the intended recipient, such as autologous cells or cells prepared from cells that are cytostatically compatible with the intended recipient. As a further example, dedifferentiated stem cells can be generated by reprogramming somatic cells or other cells by contacting the cells with one or more reprogramming factors. For example, the reprogramming factor can be expressed by the cell, for example, from an exogenous nucleic acid added to the cell, or from an endogenous gene that responds to a factor that promotes or induces expression of the gene, such as a small molecule, microRNA, or the like. The reprogramming factor can be added to an exogenous source, e.g., culture medium, and into cells by methods known in the art such as cell transduction peptides, protein or nucleic acid transfection agents, coupling with lipofectin, electroporation, gene guns, microinjection methods. It may be provided by being introduced by or the like. Factors used to reprogram somatic cells into dedifferentiated stem cells include, for example, one or more combinations selected from the group consisting of Oct4 (also referred to as Oct 3/4), Sox2, c-Myc, and Klf4. In addition, factors used to reprogram somatic cells into dedifferentiated stem cells include, for example, combinations of one or more selected from the group consisting of Oct-4, Sox2, Nanog, and Lin28. Somatic cells can be reprogrammed by expressing at least two reprogramming factors, at least three reprogramming factors, or four reprogramming factors. Certain dedifferentiated stem cell lines may have various expression profiles, but dedifferentiated stem cells can usually be identified by expression of the same markers as embryonic stem cells. Somatic cells are reprogrammed by expressing at least 1, 2, 3, 4, 5 reprogramming factors. The reprogramming factor can be selected from Oct 3/4, Sox2, NANOG, Lin28, c-Myc and Klf4. Expression of the reprogramming factor can be induced by contacting somatic cells with at least one substance, such as a small organic molecular substance, that induces the expression of the reprogramming factor.

In the present invention, "organoid" refers to a cell having a 3D three-dimensional structure, and refers to a model similar to a nerve and intestine manufactured through an artificial culture process that is not collected or acquired in an animal or the like. The origin of the cells constituting it is not limited. The organoid may have an environment allowed to interact with the surrounding environment during the cell growth process. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro. Accordingly, the 3D organoid in the present invention can be an excellent model for observing the development of therapeutic agents for diseases and the like by almost completely simulating the organs that are actually interacting in vivo.

In the present invention, "beta-amyloid" is known as a peptide consisting of 40 to 42 amino acids produced by the action of a protease from an amyloid precursor protein (APP) as a major constituent of amyloid.

In the present invention, "beta-amyloid oligomer" refers to a complex formed by integration of an amyloid precursor protein into beta-amyloid 40 or 42 (Aβ40 or Aβ monomer) by a beta-degrading enzyme. For example, it may be an oligomer containing at least one of beta-amyloid 40 oligomer, beta-amyloid 42 oligomer, and beta-amyloid 40 oligomer and beta-amyloid 42 oligomer. Among these various stages of beta-amyloid, from the state of becoming an oligomer, it exhibits strong neurotoxicity to the brain, thereby killing brain cells.

In one embodiment of the present invention, the brain organoid of step (a) ^{may be produced using 9 × 10 3} to 1 × 10 ⁴ human dedifferentiated stem cells, but is not limited thereto.

In another embodiment of the present invention, the amyloid beta oligomer of step (b) may be treated at a concentration of 8 to 12 μM for 2 to 4 days, preferably at a concentration of 10 μM for 3 days. .

In another embodiment of the present invention, the candidate stem cells in step (b) may be co-cultured with the brain organoids ^{of 1 × 10 4} to 5 × 10 ^{4 cells, but the} The number of cells may vary depending on the number of cells used to produce brain organoids, and an appropriate number of stem cells may be selected by a person of ordinary skill in the art.

In another embodiment of the present invention, the candidate stem cells in step (b) may be derived from different donors, and more specifically, stem cells derived from the same tissue derived from different donors. For example, it is possible to compare the therapeutic effect of mesenchymal stem cells derived from inferior turbinates derived from one donor and mesenchymal stem cells derived from inferior turbinates derived from another donor using the stem cell screening method according to the present invention. In addition, the candidate stem cells may be stem cells derived from different tissues of the same donor. For example, it is possible to compare the therapeutic effect between the mesenchymal stem cells derived from the lower turbinate and the mesenchymal stem cells derived from the bone marrow derived from the same donor using the stem cell screening method according to the present invention.

In addition, in one embodiment of the present invention, step (c) is a step of selecting stem cells by evaluating a therapeutic effect, and as a method of evaluating a therapeutic effect, apoptosis of nerve cells is evaluated to determine the death of nerve cells. Stem cells from the least-prone group can be selected. The neuronal cell death evaluation may be performed by a method known in the art, for example, apoptosis evaluation using the TUNEL assay. In addition, by evaluating the expression level of tubulin-beta III or nestin, which is a neuronal marker, the stem cells of the group having the least decreased expression can be selected. In addition to the above-listed methods of evaluating the therapeutic effect, methods known in the art may be appropriately selected to evaluate the therapeutic effect of stem cells or the protective effect of neurons.Therefore, the evaluation method is exemplary, and the present invention evaluates the therapeutic effect. It is not limited by the method.

In another embodiment of the present invention, the cranial nervous system disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, Batten disease, It may be one or more selected from the group consisting of cerebral infarction, cerebral hemorrhage, and spinal cord injury, but is not limited to the above-listed diseases as long as it is a cranial nervous system disease that can be improved by stem cell therapy.

The present invention can also provide a method for screening stem cells for treatment of cranial and nervous system diseases, including the following steps.

(a) preparing a three-dimensional brain organoid using human dedifferentiated stem cells;

(b) culturing the brain organoids;

(c) treating and culturing the brain organoids with amyloid beta oligomers and candidate stem cells at the same time; And

(d) selecting a stem cell having a therapeutic effect from among the candidate stem cells.

In one embodiment of the present invention, the culture of step (b) may be cultured in a medium containing mTeSR1, N2 supplement, and MEM NEAA (Non-Essential Amino Acid), preferably mTeSR1, N2 Supplement, MEM NEAA and can be cultured in a medium containing heparin.

Detailed description of the “mTeSR1” medium in the present invention is “Ludwig, T.E. et al., Nature methods 3:637-646 (2006)” and can be purchased from STEMCELL Technologies (Cat. #85850, #85857, etc.). In addition, the "N2 supplement" may be purchased from Thermo Fisher Scientific (catalog number 17502048, etc.), STEMCELL Technologies (catalog number 07152, 07156, etc.), R&D Systems (catalog number #AR033), and the like. In addition, the MEM NEAA can be purchased from Thermo Fisher Scientific (catalog number 11140050, 11140076, etc.), STEMCELL Technologies (catalog number #36550, etc.), LONZA (catalog number 13-114E, etc.).

In another embodiment of the present invention, the cultivation of step (b) may be performed for 30 to 50 days, preferably 30 to 40 days.

In addition, the present invention can provide a stem cell screening method for the treatment of neurological disorders, including the following steps.

(a) preparing a three-dimensional brain organoid using human dedifferentiated stem cells;

(b) culturing the brain organoids;

(c) treating and culturing the brain organoids with amyloid beta oligomers and candidate stem cells at the same time;

(d) selecting stem cells having a therapeutic effect from among the candidate stem cells; And

(e) comparing the treatment effect by treating the selected stem cells to a cranial nerve disease model animal.

In one embodiment of the present invention, the stem cell screening method further comprises the step of finally selecting stem cells having a higher therapeutic effect compared to other candidate stem cells in both the (d) and (e) steps. I can. Stem cells having a higher therapeutic effect may be those having a therapeutic effect of 10% to 30% compared to other candidate stem cells in steps (d) and (e).

The terms or words used in the specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventor may appropriately define the concept of terms in order to describe his own invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

[Example]

Example 1: Preparation of 3D brain organoid using human dedifferentiated stem cells

^{After mixing 9 × 10 3} or 1 × 10 ⁴ dedifferentiated stem cells derived from human blood with mTeSR, dispensing each into a U bottom 96-well plate and incubating in an environment of _{37°C and 5% CO 2} In about 7 days, spheroids were induced. Thereafter, the formed spheroids were differentiated for about 5 to 7 days using a differentiation medium containing N2 supplement and MEM NEAA. The medium was changed every 3 to 4 days until complete maturity was reached, and a shaker was put into the incubator and incubated for about 30 to 40 days in an environment of _{37° C. and 5% CO 2 at a speed of 70 rpm.}

Example 2: Using three-dimensional brain organoids in vitro Establishing Alzheimer's disease model

After incubating the 3D brain organoid prepared in Example 1 for _{about 40 days in an incubator under an environment of 37°C and 5% CO 2} , 10 μM amyloid β oligomers (Aβ) were treated, and Transwell ^® system Using, the experimental group was co-cultured for 6 days in the upper chamber in which the lower turbinate-derived mesenchymal stem cells were plated, and the control group was the upper chamber containing only medium.

As a result, as shown in Figure 1, in the organoid treated with the amyloid beta oligomer, apoptosis of nerve cells was greatly induced, and accordingly, it was confirmed that the shape of the organoid was largely changed as the organoid was released. On the other hand, it was confirmed that the organoid shape was well maintained without significant change in the organoids treated with stem cells. From the above results, it was found that the mesenchymal stem cells derived from the lower turbinate exhibit a neuroprotective effect by inhibiting neurotoxin induced by amyloid beta.

In comparison, after treatment with 10 μM amyloid beta oligomer, ^{using the Transwell ®} system, the experimental group was placed in an upper chamber plated with mesenchymal stem cells derived from inferior turbinate (hNTSCs) or bone marrow derived mesenchymal stem cells (hBMSCs) , In the case of co-culture of the upper chamber containing only the medium for the control group for 3 days, it was observed that nerve cell death was induced in the organoid treated with amyloid beta, and part of the organoid morphology was changed, but for 6 days. As in the case of culture, the phenomenon that the organoids were released could not be observed. Furthermore, in the group treated with the stem cells, it was confirmed that cell death was suppressed and the shape of the organoid was well maintained.

Through several replicates, it was found that the brain organoid morphology was much more stable when cultured for 3 days after amyloid beta treatment. Accordingly, in order to prepare and standardize a reproducible in vitro Alzheimer's disease model, it was confirmed that it is good to evaluate 10 μM amyloid beta oligomer in an _{incubator at 37° C. and 5% CO 2 in an incubator for 3 days.}

Example 3: Evaluation of treatment efficacy of stem cells using three-dimensional brain organoids

Three-dimensional brain organoids prepared using human dedifferentiated stem cells _{were incubated for about 40 days in an incubator under an environment of 37°C and 5% CO 2} , treated with 10 μM amyloid beta oligomer, and then treated with a Transwell ^® system. The upper chambers plated with turbinate-derived mesenchymal stem cells were co-cultured for 3 days, respectively.

After the cultivation was completed, it was washed once with 1X PBS (Phosphate-buffered saline), fixed with 4% paraformaldehyde for 24 hours, and then 15% and 30% sucrose solutions were sequentially treated. After that, a block was made using an OCT (Optimal cutting temperature) compound, and a frozen slide was produced. The section was washed with PBS and then treated with 0.1% Triton X-100 to permeabilize (permeabilize the cell membrane so that it can be stained). After washing again with PBS, 1% 　normal goat serum was treated at room temperature for 1 hour to block non-specific binding, and then the primary antibody against neuronal cell-related proteins (tubulin beta III or Iba- 1) was treated and reacted at 4° C. for 24 hours. Then, it was washed with PBS, treated with a secondary antibody (Alexa Fluor 594-conjugates) with fluorescence against the primary antibody, and reacted at room temperature for 1 hour. The reaction product was washed with PBS, mounted with a mounting solution, and then analyzed for expression of neuron-related proteins using confocal microscopy by immunofluorescence staining. DAPI (4',6-diamidino-2-phenylindole) was used for counterstaining. In addition, an analysis of the degree of cell death was performed through TUNEL staining.

Example 4: Evaluation of the therapeutic efficacy of stem cells according to donor and origin

As described in Example 3, 3D brain organoids prepared using human dedifferentiated stem cells _{were cultured in an incubator under an environment of 37°C and 5% CO 2} for about 40 days and treated with 10 μM amyloid beta oligomer. After that, the upper chamber in which the inferior turbinate-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells obtained from two different donors were plated, respectively, was co-cultured for 3 days using the ^{Transwell ® system.} . After the cultivation was completed, after washing with 1X PBS and fixing with 4% paraformaldehyde for 24 hours, a block was manufactured using an OCT compound through 15% and 30% sucrose treatment. After preparing the frozen section, it was washed with PBS, and the whole organoid shape was observed by H&E (Hematoxylin & Eosin) staining.

As a result, as shown in Figure 3, in the organoid treated with amyloid beta, it was confirmed that the death of nerve cells was greatly induced, and a part of the organoid was damaged. On the other hand, in the group treated with stem cells together, the degree of tissue damage to organoids was less than in the group treated with only amyloid beta.

Furthermore, the effect differed according to the donor of stem cells and the type of tissue derived from it. That is, as shown in FIG. 3, it was confirmed that in the case of the lower turbinate-derived stem cells, the morphology was better maintained in the organoid treated with the stem cells of the 376 donor than the stem cells of the 473 donor. It was confirmed that it was larger when the inferior turbinate-derived stem cells were treated than when the derived stem cells were treated. Therefore, the present inventors determined that the treatment efficiency of stem cells derived from inferior turbinate of donor 376 is the best.

Example 5: Evaluation of treatment efficacy of stem cells through analysis of protein expression related to nerve cells

As described in Example 4, the three-dimensional brain organoids produced using human dedifferentiated stem cells _{were cultured in an incubator under an environment of 37° C. and 5% CO 2} for about 40 days and treated with 10 μM amyloid beta oligomer. a rear, a top chamber in the Transwell ^® using a system of two mutually inferior turbinate-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells from different donors are plated each were co-cultured for 3 days. After the cultivation was completed, it was washed once with 1X PBS, fixed with 4% paraformaldehyde for 24 hours, and then subjected to 15% and 30% sucrose treatment to prepare a block using an OCT compound. Frozen sections were prepared, washed with PBS, and permeabilized using 0.1% Triton X-100. After washing again with PBS, 1% normal goat serum was treated at room temperature for 1 hour to block non-specific binding, and then treated with a primary antibody (tubulin beta III or Iba-1) against neuronal cell-related proteins and treated at 4°C for 24 hours. It was allowed to react for hours. Then, it was washed with PBS, treated with a secondary antibody (Alexa Fluor 594-conjugates) with fluorescence against the primary antibody, and reacted at room temperature for 1 hour. The reaction product was washed with PBS, mounted with a mounting solution, and then analyzed for expression of neuron-related proteins using confocal microscopy by immunofluorescence staining. DAPI (4',6-diamidino-2-phenylindole) was used for counterstaining.

As a result, as shown in FIG. 4, it was confirmed that neuron expression was much greater in organoids treated with stem cells derived from inferior turbinate tissue compared to organoids treated with bone marrow-derived stem cells together.

Example 6: Evaluation of treatment efficacy of stem cells through cytokine analysis

After the human de-differentiation produced by the stem cells three-dimensional brain organosilane Carcinoid the 37 ℃, 5% CO ₂ environment and culture medium cultured for about 40 days and handle 10 μM beta oligomers, by using the Transwell ^® system 2 Inferior turbinate-derived mesenchymal stem cells or bone marrow-derived mesenchymal stem cells obtained from different donors were plated in the upper chamber for 3 days. After the culture was completed, the culture medium was collected and analyzed for differences in proteins secreted from organoids through cytokine assay.

As a result, as shown in FIG. 5, when compared with the organoids of the group treated with only amyloid beta, the organoids of the group treated with stem cells also expressed various proteins such as TIMP1, TIMP2, OPN, OPG, and IL-6. It was confirmed that a difference appeared.

Example 7: in vivo Through experiment in vitro Validation of the screening platform

The present inventors used the Alzheimer's animal model to confirm the therapeutic effect of the stem cells identified through the above examples in the same manner in vivo , and the therapeutic effect of the lower turbinate-derived mesenchymal stem cells and the bone marrow-derived mesenchymal stem cells Was confirmed.

As a result, as shown in Figs. 6a to 6c, in the group treated with the inferior turbinate-derived mesenchymal stem cells of donor 376, the expression of amyloid beta was most suppressed, and the expression ratio of TIMP-2 was increased. It was confirmed that the improvement was larger. These results are consistent with the results of the treatment effect of stem cells identified through the above examples, and prove that the stem cell screening method according to the present invention is effective.

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and are not limiting.

Claims (13)

Stem cell screening method for treating cranial nerve system diseases, comprising the following steps:
(a) preparing a 3-Dimensional brain organoid using human dedifferentiated stem cells;
(b) treating and culturing the brain organoids with amyloid beta oligomers and candidate stem cells at the same time; And
(c) selecting a stem cell having a therapeutic effect from among the candidate stem cells.
The method of claim 1,
The three-dimensional brain organoid of step (a) ^{is characterized in that produced using 9 × 10 3} to 1 × 10 ⁴ human dedifferentiated stem cells, stem cell screening method.
The method of claim 1,
The amyloid beta oligomer of step (b) is characterized in that it is treated for 2 to 4 days at a concentration of 8 to 12 μM, stem cell screening method.
The method of claim 1,
Candidate stem cells of the step (b) is characterized in that 1 × 10 ⁴ to 5 × 10 ⁴ cells co-cultured with the brain organoids (co-culture), stem cell screening method.
The method of claim 1,
The stem cell screening method, characterized in that the candidate stem cells in step (b) are derived from different donors.
The method of claim 1,
The stem cell screening method, characterized in that the candidate stem cells in step (b) are derived from different types of tissues of the same donor.
The method of claim 1,
In the step (c), the stem cells of the group with the least decrease in neuronal cell death, the group with the least decrease in the expression of tubulin-beta III, or the group with the least decrease in the expression of nestin. A method for screening stem cells, characterized in that selection.
The method of claim 1,
The cranial nervous system diseases consist of Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, Batten disease, cerebral infarction, cerebral hemorrhage and spinal cord injury. It is characterized in that at least one selected from the group, stem cell screening method.
Stem cell screening method for treating cranial nerve system diseases, comprising the following steps:
(a) preparing a three-dimensional brain organoid using human dedifferentiated stem cells;
(b) culturing the brain organoids;
(c) treating and culturing the brain organoids with amyloid beta oligomer and candidate stem cells at the same time; And
(d) selecting a stem cell having a therapeutic effect from among the candidate stem cells.
The method of claim 9,
The culture of step (b) is characterized in that the culture is cultured in a medium containing mTeSR1, N2 supplement, and MEM NEAA (Non-Essential Amino Acid), stem cell screening method.
The method of claim 9,
The cultivation of step (b) is characterized in that made for 30 to 40 days, stem cell screening method.
Stem cell screening method for treating cranial nerve system diseases, comprising the following steps:
(a) preparing a three-dimensional brain organoid using human dedifferentiated stem cells;
(b) culturing the brain organoids;
(c) treating and culturing the brain organoids with amyloid beta oligomer and candidate stem cells at the same time;
(d) selecting stem cells having a therapeutic effect from among the candidate stem cells; And
(e) comparing the treatment effect by treating the selected stem cells to a cranial nerve disease model animal.
The method of claim 12,
The stem cell screening method further comprising the step of finally selecting stem cells having a higher therapeutic effect compared to other candidate stem cells in both of the (d) and (e) steps.

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