WO2018056616A1 - Method for screening patient-specific stem cell therapeutic agent - Google Patents

Abstract

The present invention relates to a method for screening a patient-specific therapeutic agent. When using the method for screening a patient-specific therapeutic agent of the present invention, it is possible to identify and select the therapeutic ability of mesenchymal stem cells of different origins with respect to disease subtypes or disease effectors. In addition, it is possible to confirm the characteristics of the secretory protein which acts at high efficiency or low efficiency, by analyzing proteins secreted by the selected mesenchymal stem cells, and thus there is the advantage that a customized therapeutic agent for the specific nature of the disease of each patient can be proposed.

Description

Screening method of patient-specific stem cell therapy

The present invention relates to a method for screening a patient-specific stem cell therapy.

Although various therapeutic agents have been developed for treating diseases, the disease-causing cells having various kinds of genetic characteristics are present in the same disease, and even if the patient has the same disease, Suitable therapeutic agents may be different.

Even if one treatment kills certain diseased cells and does not kill all the disease-causing cells with other genetic characteristics, a treatment that is appropriate for one patient may not be effective in another. Rather, it may cause adverse effects that aggravate the disease.

In order to solve this problem, various drugs have been developed to study and treat various disease-causing mechanisms.However, a separate standard method for determining whether a drug can be used to achieve a desired therapeutic effect As it is not reported, there is a limit to use as a therapeutic agent.

Therefore, even if the patient's disease is the same, the disease-causing variation may vary from patient to patient, so selecting a therapeutic agent suitable for a patient-specific mutation is a very important problem, and securing a method for screening a patient-specific treatment agent. Is needed.

When the inventors of the present invention are studying a method for selecting a patient-specific customized therapeutic agent, co-culturing a patient-derived cell and a candidate group of mesenchymal stem cells, and confirming the change of the patient-derived cell according to the co-culture. The present invention was completed by confirming that stem cells showing a particularly excellent effect on patients or diseases can be selected quickly.

Accordingly, an object of the present invention is to provide a method for screening a patient-specific stem cell therapy.

In order to solve the above problems, the present invention comprises the steps of (1) co-culturing the patient-derived cells and candidate stem cells; And (2) selecting stem cells by measuring changes in proliferative, differentiating or pathological markers of patient derived cells; It provides a method for screening a patient-specific stem cell therapy comprising a.

When using the screening method of the patient-specific stem cell therapeutic agent of the present invention, it is possible to confirm and select the therapeutic ability of mesenchymal stem cells of different origin for the disease subtype or disease effector. In addition, by analyzing the proteins secreted by the selected mesenchymal stem cells can determine the properties of the secreted proteins that work in a high efficiency or low efficiency, there is an advantage that can propose a tailored therapeutic agent for disease specificity of each patient.

1 is a schematic diagram showing a co-culture method of a screening model for selecting stem cells showing an excellent therapeutic effect on Alzheimer's dementia (AD) neuron cells.

Figure 2 is a cord blood-derived mesenchymal stem cells (UCB-MSC), umbilical cord-derived mesenchymal stem cells (WJ-MSC) or fat-derived mesenchymal stem cells (Adipose-MSC) in the treatment group to check the degree of cell activity to prevent cell death The figure which showed the result which confirmed the effect.

Figure 3 is a diagram showing the results of the classification of secretion proteins by function of umbilical cord-derived mesenchymal stem cells (WJ-MSC) treatment group.

Figure 4 is a diagram showing the results of the classification of secreted proteins by the function of adipose-derived mesenchymal stem cells (Adipose-MSC) treatment group.

 5 is a diagram showing a method for establishing a stem cell therapeutic screening model for each Alzheimer's type dementia subtype.

Figure 6 shows the expression level of SOX2 and Nestin of APP-NPC or PS-1-NPC in adipose-derived mesenchymal stem cells (Adipose-MSC) treatment group or umbilical cord-derived mesenchymal stem cell (WJ-MSC) treatment group Is a diagram showing.

7 shows MAP2 and Tuj1 (Neuron-specific class III) of APP-NPC or PS-1-NPC in adipose-derived mesenchymal stem cell (Adipose-MSC) treated group or umbilical cord-derived mesenchymal stem cell (WJ-MSC) treated group. The figure which showed the result of having confirmed the expression level of beta-tubulin).

Figure 8 shows the co-culture of Alzheimer subtype-derived PS1-NPC, sAD (Sporadic AD) -NPC and fat-derived mesenchymal stem cells (Adipose-MSC) treatment group or umbilical cord-derived mesenchymal stem cell (WJ-MSC) treatment group It is a figure which shows the result of comparing cell proliferation ability.

Figure 9 compares the cell proliferation capacity of co-culture of Alzheimer subtype-derived PS1-NPC, sAD-NPC and adipose-derived mesenchymal stem cell (Adipose-MSC) treatment group or umbilical cord-derived mesenchymal stem cell (WJ-MSC) treatment group Figure 1 shows the results.

FIG. 10 shows the pathological factor ubiquitin according to co-culture of Alzheimer subtype-derived PS1-NPC, sAD-NPC and adipose-derived mesenchymal stem cell (Adipose-MSC) treated group or umbilical cord-derived mesenchymal stem cell (WJ-MSC) treated group. It is a figure which shows the result of comparing and quantifying the change of expression of a conjugate.

11 is a diagram showing a method for establishing a stem cell therapeutic screening model using neurons derived from Alzheimer's disease patients.

Figure 12 compares and quantified the expression changes of ubiquitin conjugates according to co-culture of Alzheimer's patients-derived neurons and adipose-derived mesenchymal stem cells (Adipose-MSC) treatment group or umbilical cord-derived mesenchymal stem cell (WJ-MSC) treatment group. The results are shown.

The present invention comprises the steps of (1) co-culturing the patient-derived cells and candidate stem cells; And (2) selecting stem cells by measuring changes in proliferative, differentiating or pathological markers of patient derived cells; It provides a method for screening a patient-specific stem cell therapy comprising a.

The screening method of the present invention enables the rapid selection of stem cells that can have the most effect on patient cells, and thus can be effectively used for screening patient-specific stem cell therapeutics and developing them as therapeutic agents. Hereinafter, the present invention will be described in detail.

Step (1) of the present invention is a step of co-culturing patient derived cells and candidate stem cells.

In the present invention, "patient-derived cell" means a cell isolated and obtained from a patient, and since the cell is directly isolated from the patient, a cell having a characteristic capable of reflecting all genetic and pathological factors of the individual patient. it means. Therefore, it can be expected that the selected stem cell therapeutics that have an effect on the patient-derived cells will have an excellent effect on the patient.

The patient-derived cell may be a cell derived from a patient having each disease subtype, and when used, it is possible to effectively select a stem cell therapeutic agent having a particular effect on a specific disease subtype.

The patient-derived cells of the present invention may preferably be cells derived from a patient with degenerative brain disease, and the cells derived from the patient with degenerative brain disease are derived differentiated stem cells derived from patients with degenerative brain disease or subtypes thereof (Induced Pluripotent Stem Cell, iPSC), nerve cell or nerve progenitor cell nerve.

In the present invention, the "degenerative brain disease" is a specific brain cell group of the brain and spinal cord gradually loses its function, the death of cerebral neurons which is most important for information transmission of the nervous system, synapses that transmit information between the brain neurons and brain nerve cells It is a disease caused by the formation or function problems of the brain and the ideal increase or decrease of electrical activity of the cranial nerve. Degenerative brain diseases are classified by considering the main symptoms and invading brain areas, and include Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington's disease (HD).

Therefore, the degenerative brain disease of the present invention may be one or more selected from the group consisting of forgetfulness, dementia, Alzheimer's disease, Parkinson's disease and Huntington's disease.

In the present invention, the "candidate stem cell" means a stem cell population used for the purpose of selecting stem cells showing the best effect. Among the candidate stem cells, cells showing the best effect on the patient-derived cells as compared to other stem cells can be selected as a patient-specific stem cell therapy.

Candidate stem cells of step (1) may be derived from one or more selected from the group consisting of cord blood, umbilical cord, bone marrow and fat. For the purposes of the present invention, the stem cells may be mesenchymal stem cells. According to the screening method of the present invention, it is possible to select a stem cell showing the most excellent effect on the patient-specific, subtype-specific. In one embodiment of the present invention using the cells derived from Alzheimer's patient co-culture with the candidate stem cells, the effect of each candidate stem cells on Alzheimer's patients derived neuro precursor cells, neurons, and improve the pathological state of Alzheimer's To this end, umbilical cord-derived mesenchymal stem cells are appropriate, and for the purpose of proliferation of neurons, it was confirmed that it is preferable to select umbilical cord-derived mesenchymal stem cells rather than fat-derived mesenchymal stem cells.

Co-culture of step (1) of the present invention can be made using a transwell. For example, one embodiment of the present invention provides a method of inoculating a patient-derived cell on top of a chamber of a transwell and inoculating candidate stem cells on a bottom of a transwell chamber. In this culture, by the interaction of the patient-derived cells and the candidate stem cells, the candidate stem cells can secrete a variety of substances that can affect the patient-derived cells, thereby increasing the growth of the cells derived from the patient May be promoted, expression of pathological markers may be reduced, or cell differentiation may be promoted.

The term "culture" in the present invention means to grow the cells under environmental conditions that are appropriately artificially controlled, preferably culture may be carried out in the transwell.

The patient derived cells and candidate stem cells can be grown in a conventional medium. The medium may contain a nutritional substance required by the cells to be cultured, that is, the cells to be cultured in order to culture specific cells, and may be mixed with an additional material for a special purpose. The medium may also be referred to as an incubator or a culture medium, and is a concept that includes all natural, synthetic, or selective media. The patient-derived cells and candidate stem cells can be cultured according to conventional culture methods.

The co-culture of step (1) may be performed for about 5 to 14 days, preferably 5 to 10 days, more preferably 5 to 7 days. At this time, it is preferable to replace the culture medium every two to three days.

Step (2) of the present invention is a step of selecting mesenchymal stem cells by measuring the change in proliferative capacity, differentiation capacity or pathological markers of patient-derived cells.

In the selection of step (2), stem cells capable of significantly inducing a desired change than other candidate stem cells by measuring a change in proliferative capacity, differentiation capacity or pathological markers of a patient-derived cell are stemmed according to a patient or disease subtype. Selection as a cell therapeutic agent, and the desired change may include without limitation changes that promote cell proliferation, promote differentiation, or inhibit the expression of pathological markers.

When the patient-derived cells are degenerative brain disease-derived cells, the proliferative capacity of step (2) can be confirmed by measuring the survival rate of the cells derived from the patients with degenerative brain disease, and the differentiation capacity is MAP2 (microtubule associated protein 2), NF-200 ( neurofilament 200), Tuj1 (Neuron-specific class III beta-tubulin) and VGLUT2 (vesicular glutamate transporter 2) can be characterized by measuring the expression of one or more neuronal differentiation markers selected from the group consisting of.

In addition, the change in the pathological marker of step (2) may be characterized by measuring the expression of one or more pathological markers selected from the group consisting of ubiquitin conjugates, amyloid beta and tau proteins, inflammation-related cytokines and the like.

In another aspect, the present invention may further comprise the step of analyzing the secreted proteins of the selected stem cells of the step (2), after the selection step of (2).

For the purposes of the present invention, the secreted proteins of the stem cells can be classified by function to select specifically expressed secreted proteins. For example, if stem cells are selected that significantly promote the proliferation of patient-derived cells when co-cultured with patient-derived cells among candidate stem cells, the proteins secreted from the selected stem cells are concentrated and analyzed to significantly promote proliferation of certain proteins. You can see if you can.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example  1. Effects on Alzheimer's Dementia Mesenchyme  Stem Cell Screening Model

1-1. Screening model for the identification of mesenchymal stem cells for Alzheimer's dementia (AD)

A screening model was constructed to compare the therapeutic potential of various mesenchymal stem cells (MSCs) against Alzheimer's disease (AD). Specifically, as shown in Figure 1, HT22, a hippocampal neuronal cell line of the mouse was inoculated in a medium essential medium-alpha (MEM-alpha) medium in the bottom of the transwell. Thereafter, amyloid beta 42, an Alzheimer's type dementia-specific biomarker and a toxic molecule, was added to oligomer form in the lower well of the transwell inoculated with the HT22 cells to induce cell death. Alzheimer's dementia model was constructed. Then, coculture was performed by inoculating cord blood-derived mesenchymal stem cells (UCB-MSC), umbilical cord-derived mesenchymal stem cells (WJ-MSC), or adipose-derived mesenchymal stem cells (Adipose-MSC), respectively. . A group not inoculated with MSC was set as a control group, and the effect of preventing cell death by MSCs derived from the above-mentioned different species was confirmed. Specifically, the expression level of the differentiation marker MAP2 was confirmed through fluorescence. MAP2 protein is a protein that extends to the nerve cell ends during neuronal differentiation, and may be an indicator of whether the cell ends are damaged by neuronal cell death. Therefore, the fluorescence measurement of MAP2 can confirm whether the cells are killed through co-culture of MSC, the results are shown in FIG.

As shown in FIG. 2, the group inoculated and co-cultured with cord blood-derived mesenchymal stem cells (UCB-MSC), umbilical cord-derived mesenchymal stem cells (WJ-MSC) or adipose-derived mesenchymal stem cells (Adipose-MSC), respectively. It was confirmed that the increase in fluorescence expression of MAP2 and the effect of preventing apoptosis appeared to be effective for treating Alzheimer's dementia, thereby confirming the therapeutic ability of various mesenchymal stem cells, and candidate mesenchymal stem cell group It was confirmed that the effect of can be compared and analyzed.

1-2. Identification of Secretory Proteins of Mesenchymal Stem Cells Associated with Alzheimer's Dementia

According to the co-culture of each group of 1-1, in order to identify the protein secreted by different MSCs, cord blood-derived mesenchymal stem cells (UCB-MSC), umbilical cord-derived mesenchymal stem cells (WJ-MSC) Alternatively, the culture medium at the bottom of the transwell of the adipose-derived mesenchymal stem cell (Adipose-MSC) treated group was collected, and the collected culture solution was concentrated to increase the concentration of the secreted protein. Thereafter, antibody arrays were performed for the culture medium of each of the concentrated groups. Thereafter, the array results were identified using an analysis program to identify proteins showing more than twofold fold changes in each source. Thereafter, neuronal proteins were separately classified among secretory proteins of umbilical cord-derived mesenchymal stem cells (WJ-MSC) or adipose-derived mesenchymal stem cells (Adipose-MSC) treated groups. Thereafter, the neuronal proteins were reclassified by function to confirm the characteristics of the neuronal proteins secreted by the treatment groups. The results are shown in FIGS. 3 and 4.

As shown in Figures 3 and 4, the umbilical cord-derived mesenchymal stem cells (WJ-MSC) treated group and adipose derived mesenchymal stem cells (Adipose-MSC) treated group showed different protein secretion characteristics. As shown in FIG. 3, umbilical cord-derived mesenchymal stem cells (WJ-MSCs) secrete more secretory proteins such as nervous system development, neuronal production, and axon guidance, and secrete more secretory proteins related to the survival of neurons. It was. In addition, as shown in Figure 4, Adipose-derived mesenchymal stem cells (Adipose-MSC) treated group secreted more proteins involved in the development of the nervous system, signaling pathways, neuronal cell production, cell survival and proliferation It was confirmed that a lot of related proteins secreted.

These results indicate that even mesenchymal stem cells may be secreted from different populations of secreted proteins for the treatment of diseases, and thus, specific mesenchymal stem cells can be screened for screening patient-specific therapeutic agents. . For example, in order to select tailored MSC therapies that can alter Alzheimer's dementia factors, co-cultivation of MSCs of different origin and patient-derived disease cells can reduce disease markers or prevent neuronal cell death. MSCs can be screened. In addition, the proteins secreted by different MSCs can be identified to select proteins that have an effect on Alzheimer's dementia.

Example 2 Screening by Subtypes of Alzheimer's Dementia Using Mesenchymal Stem Cells

Based on the screening model of Example 1, in order to build a subtype-specific screening model of the Korean type Alzheimer's dementia, the mesenchymal stem cells of the patient-derived neuronal progenitor cells (NPC) of the Korean type Alzheimer's type dementia The therapeutic ability was confirmed.

Specifically, based on the screening model of Example 1, as shown in FIG. 5, NPCs (AD-NPC) and MSCs derived from Alzheimer's patient were co-cultured and MSCs having a therapeutic effect were selected. The bottom of the trans well was first coated with poly-L-ornithine (PLO) and laminin. Thereafter, neuroprogenitor APP-NPC or PS-1-NPC of a patient corresponding to the Korean type Alzheimer's dementia subtype APP or PS-1 was inoculated at the bottom of the transwell at ^2.5 × 10 ⁴ / cm ² . The neural progenitor cells are patient specific cells prepared by collecting somatic cells from dementia patients who visited Samsung Medical Center and dedifferentiating them. Umbilical cord-derived mesenchymal stem cells (WJ-MSC) or adipose-derived mesenchymal stem cells (Adipose-MSC) were inoculated into the insert chamber, respectively, and the ratio of AD-NPC and MSC was 10: 3. After inoculating APP-NPC or PS-1-NPC for each subtype and confirming its culture state one day later, in differentiation medium (Neurobasal A media + B27 w / o vit.A + Glutamax + ｐ / s + BDNF + NT3) Co-culture was carried out for 7 days. Thereafter, the stem cell capacity, differentiation capacity and pathological markers of the umbilical cord-derived mesenchymal stem cells (WJ-MSC) or adipose-derived mesenchymal stem cells (Adipose-MSC) were identified. Stem cell capacity and differentiation capacity were confirmed by Western blot and immunocytochemistry for the expression level of SOX2 / Nestin marker and MAP2 / Tuj1 in AD-NPC co-cultured for 1 week. The results are shown in FIGS. 6 and 7.

As shown in Figures 6 and 7, umbilical cord-derived mesenchymal stem cells (WJ-MSC) and adipose-derived mesenchymal stem cells (Adipose-MSC) were confirmed to show different effects on subtypes, respectively.

Example 3. Screening of Stem Cell Therapeutics Using Patient-Derived Neural Precursor Cells

For tailored mesenchymal stem cell screening for patient-derived NPCs, sAD (sporadic AD) -NPC or PS-1-NPC and umbilical cord-derived mesenchymal stem cells (WJ) corresponding to Alzheimer's dementia sAD or PS-1 -MSC) or adipose-derived mesenchymal stem cells (Adipose-MSC) were each co-cultured, and the effect of each stem cell on NPC was confirmed. The experiment was carried out in the same manner as in Example 2.

3.1 Mesenchymal Stem Cell Screening with Excellent Proliferation Promoting Activity-CCK-8 Assay

CCK-8 cell viability assay was performed for 48 hours to confirm the effect of promoting patient-derived NPC proliferation by umbilical cord-derived mesenchymal stem cells (WJ-MSC) or adipose-derived mesenchymal stem cells (Adipose-MSC). Was confirmed through micrographs and CCK absorbance changes. The bFGF used in the experiment is a substance added during neuronal progenitor cell culture and proliferation, and is treated to maintain the progenitor cell state by preventing neural progenitor cell differentiation. bFGF was treated to confirm the change in the state or proliferation of cells with or without bFGF. As a control group, -bFGF group was used, which is a negative control group in the experiment confirming the proliferation promoting effect when co-cultured mesenchymal stem cells. The results are shown in FIG.

As shown in FIG. 8, the proliferation of NPCs was different depending on the type of inoculated MSCs. Both sAD-NPC or PS-1-NPC increased proliferation by cord-derived mesenchymal stem cells (WJ-MSC) or adipose-derived mesenchymal stem cells (Adipose-MSC), but umbilical cord-derived mesenchymal stem cells (WJ-MSC) ) Showed more excellent cell proliferation promoting ability. Therefore, when the purpose of the proliferation of neurons, it can be interpreted that it is preferable to select and use umbilical cord-derived mesenchymal stem cells rather than fat-derived mesenchymal stem cells.

3.2 Mesenchymal Stem Cell Screening with Excellent Proliferation Promoting-BradU Assay

BradU assay was performed for 48 hours to confirm the effect of promoting NPC proliferation of the patient by umbilical cord-derived mesenchymal stem cells (WJ-MSC) or adipose-derived mesenchymal stem cells (Adipose-MSC). The control was set in the same manner as 3.1, and the results are shown in FIG. 9.

As shown in Fig. 9, the proliferation of NPCs was different depending on the type of inoculated MSCs. In particular, it was confirmed that PS-1-NPC significantly increased proliferation by umbilical cord-derived mesenchymal stem cells (WJ-MSC) and adipose-derived mesenchymal stem cells (Adipose-MSC). In the case of sAD-NPC, cell proliferation was promoted by umbilical cord-derived mesenchymal stem cells (WJ-MSC), but this effect was not confirmed in co-culture with adipose-derived mesenchymal stem cells (Adipose-MSC). BruU experiments use the principle of intercalation between DNA when cells divide and proliferate, thus eliminating the interference of metabolism of the cell itself in addition to proliferation, thus enabling more accurate screening of the cell. Therefore, in the case of the purpose of the proliferation of neurons, it can be interpreted that it is preferable to select the umbilical cord-derived mesenchymal stem cells rather than the adipose-derived mesenchymal stem cells, as well as appropriate stem cell therapy according to the disease subtype of each patient. It can be seen that there is a combination, which can be quickly confirmed by the screening method of the present invention.

3.3 Mesenchymal Stem Cell Screening to Reduce Ubiquitin Conjugates

In order to screen mesenchymal stem cells that have a pathological effect on neuroprogenitor cells, the effects of ubiquitin conjugates on the increase and decrease were identified. In neuroprogenitor cells, ubiquitin conjugates increase when abnormal intracellular substances accumulate, and ubiquitin increases when diseases such as Alzheimer's occur. Therefore, co-culture of Alzheimer's patient-derived NPCs with adipose-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells was performed by the method according to Example 2, and the increase and decrease of the ubiquitin conjugate was confirmed in the patient-derived NPCs. The increase and decrease of the ubiquitin conjugate was confirmed by Western blot, and the results are shown in FIG. 10.

As shown in Figure 10, compared to the control group without Alzheimer's disease, the ubiquitin conjugates increased in sAD, it was confirmed that the level of ubiquitin conjugates is reduced by co-culture with umbilical cord mesenchymal stem cells. In addition, the effect of reducing the ubiquitin conjugates was not found in the adipose derived mesenchymal stem cells, but rather showed an increasing aspect, confirming that it is not suitable for reducing the ubiquitin conjugates. Therefore, the mesenchymal stem cells suitable for improving the pathological condition of Alzheimer's through the screening method of the present invention can be identified and selected from the umbilical cord-derived mesenchymal stem cells.

Example 4 Screening of Stem Cell Therapeutics Using Neurons

4.1 Co-culture of Neurons and Mesenchymal Stem Cells for Screening

In order to confirm whether the customized screening according to the present invention can be applied even when using neurons (neurons) of Alzheimer's patients, NPCs were differentiated to obtain degenerative subtypes of neurons and screening using the same. The dementia subtype was a mutation of the genetic subtype APP (Amyloid Precursor Protein) / mutation of PS1 (Presenilin 1) / non-genetic subtype sAD (sporadic AD), and after the production of patient-derived dedifferentiated stem cells (iPSC), Cells differentiated by neural lineage were obtained and used for the experiment.

The screening method is similar to that of Example 2, but in order to co-culture MSC and neurons, inoculated NPCs of Korean Alzheimer's dementia subtypes at a rate of 2x10 ⁴ / cm ² and differentiated for 10 weeks to differentiate neurons by dementia subtypes. Cells were obtained. The obtained dementia subtype neurons were inoculated at the bottom of the transwell at 2.5x10 ⁴ / cm ² , and umbilical cord-derived mesenchymal stem cells (WJ-MSC) or adipose-derived mesenchymal stem cells (Adipose-MSC) were inserted into the insert chamber. Each was inoculated to be ⁴ / cm ² . Thereafter, after confirming the culture state of the neurons for 1 day, co-culture was carried out for 7 days under differentiation medium conditions. The coculture procedure used is shown in FIG. 11.

4.2 Mesenchymal Stem Cell Screening to Reduce Ubiquitin Conjugates

Co-cultivation was carried out through the method of 4.1, and mesenchymal stem cells were effectively screened and selected to effectively reduce the pathological characteristics of Alzheimer's disease through the method of 3.3. As a control group, an experimental group (B + G + N) containing only BDNF, GDNF, and NT3, which are reagents used for differentiating neurons from neuronal progenitor cells, was used. It is shown in 12.

As shown in FIG. 12, the ubiquitin conjugate was increased in the dementia patient-derived neurons, and the level of the ubiquitin conjugate was decreased by co-culture with adipose-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells. In addition, the ubiquitin conjugate reduction effect was more excellent in umbilical cord-derived mesenchymal stem cells than adipose derived mesenchymal stem cells. Therefore, the mesenchymal stem cells suitable for improving the pathological condition of Alzheimer's through the screening method of the present invention can be identified and selected from the umbilical cord-derived mesenchymal stem cells.

Through the above results, when using the screening method of the patient-specific therapeutic agent of the present invention, it was confirmed that the most suitable patient-specific stem cell therapeutics can be selected and applied for each patient's disease or subtype thereof.

Claims (10)

1. (1) co-culture of patient derived cells and candidate stem cells; And

   (2) selecting mesenchymal stem cells by measuring changes in proliferative, differentiating or pathological markers of patient derived cells; Screening method for a patient-specific stem cell therapy comprising a.
2. The method of claim 1,

   The patient-derived cells are degenerative brain disease patients derived cells, characterized in that the screening method of patient-specific stem cell therapy.
3. The method of claim 1, wherein the degenerative brain disease patient-derived cells are induced differentiated stem cells (Induced Pluripotent Stem Cells, iPSCs), neurons, or neuronal progenitor cells.
4. The method of claim 2, wherein the degenerative brain disease is one or more selected from the group consisting of forgetfulness, dementia, Alzheimer's disease, Parkinson's disease, and Huntington's disease.
5. The method of claim 1,

   The stem cell of step (1) is characterized in that derived from one or more selected from the group consisting of umbilical cord blood, umbilical cord, bone marrow and fat, screening method of patient-specific stem cell therapy.
6. According to claim 1, wherein the co-cultivation is carried out in a transwell, patient-derived cells are characterized in that the cultured by inoculating the top of the transwell chamber, the candidate stem cells inoculated in the bottom of the transwell chamber, Screening method.
7. The method of claim 2, wherein the proliferative capacity of step (2) is confirmed by measuring survival rate of cells derived from patients with degenerative brain disease.
8. The method according to claim 2, wherein the differentiation capacity of step (2) is MAP2 (microtubule associated protein 2), NF-200 (neurofilament 200), Tuj1 (Neuron-specific class III beta-tubulin) and VGLUT2 (vesicular glutamate transporter 2) The method of screening for a patient-specific stem cell therapeutic agent, characterized in that confirmed by measuring the expression of one or more neuronal differentiation markers selected from the group consisting of.
9. The method of claim 2, wherein the change of the pathological marker of step (2) is characterized by measuring the expression of one or more pathological markers selected from the group consisting of uniquitin conjugates, amyloid beta and tau protein. Screening method for customized stem cell therapy.
10. The method of claim 1,

    (3) analyzing the secreted protein of the selected stem cells of the step (2); characterized in that it further comprises, the method for screening a patient-specific stem cell therapy.

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