KR102188572B1 - Formulation for injection of stem cells containing cerebrospinal fluid and method for production - Google Patents

Abstract

It relates to a stem cell dosage form and a method of manufacturing the same, according to the stem cell dosage form and its manufacturing method according to an aspect, as well as being safe in terms of immunity, and maintaining stem cell properties and tailoring the gene or There is an effect that protein expression is changed or gene or protein expression is more activated.

Description

Formulation for injection of stem cells containing cerebrospinal fluid and method for production thereof

It relates to a stem cell administration formulation and a method of manufacturing the same.

Stem cells are cells that have the ability to differentiate into two or more cells while having self-replicating ability.Totipotent stem cells, pluripotent stem cells, and multipotent stem cells ( multipotent stem cells).

A totipotent stem cell is a cell that has the ability to develop as a complete individual. Cells up to the 8-cell stage after fertilization of eggs and sperm have these properties. When transplanted into, it can develop as a single complete entity. Pluripotent stem cells are cells that can develop into various cells and tissues derived from the ectoderm, mesoderm, and endoderm layers.The inner cell mass located inside the blastocyst appears 4-5 days after fertilization. ), which is called an embryonic stem cell, and differentiates into various other tissue cells, but cannot form new organisms. Multipotent stem cells are stem cells that can only differentiate into cells specific to the tissues and organs that contain these cells.

Stem cells, which can be obtained in large quantities, are increasingly being used in the field of medicine, including treatment of incurable diseases, as well as as a cell therapy that injects stem cells themselves for purposes such as beauty and plastic surgery.

As a conventional technique for preserving stem cells, there is a technique in which human adipose tissue-derived mesenchymal stem cells are suspended and stored in physiological saline in refrigerated conditions, but in this case, it is difficult to show a survival rate of 70% or more when stored for 48 hours or longer. In addition, in the case of the existing stem cell treatment, a culture medium or the like was used to be administered to a patient. However, it is difficult to accurately analyze the components, the proportion of additives, and the effect on cells, which may raise a problem in verifying safety.

Accordingly, as a result of the present inventors' efforts to develop a dosage form for stem cells, in the case of patient-derived body fluids, the body fluids are the same as those in the body, and in particular, the patient's body fluids are not only safe in terms of immunity, but also maintain stem cell properties. The present invention was completed by confirming that the expression of the gene or protein of the stem cell is changed or the expression of the gene or protein is further activated in a patient-tailored manner.

One aspect is to provide a stem cell dosage form comprising cerebrospinal fluid.

Another aspect is to provide the use of cerebrospinal fluid for the manufacture of a stem cell dosage form.

Another aspect is to provide a method of treating a disease comprising administering to an individual a formulation comprising cerebrospinal fluid and stem cells.

Another aspect is to provide the use of cerebrospinal fluid and stem cells for use in the manufacture of an agent for the treatment of a disease.

Another aspect is to provide a method for producing patient-specific stem cells, comprising the step of maintaining stem cells in cerebrospinal fluid, or a method for preparing a stem cell dosage form.

One aspect is to provide a stem cell dosage form comprising cerebrospinal fluid.

Another aspect is to provide the use of cerebrospinal fluid for the manufacture of a stem cell dosage form.

Specifically, the formulation is cerebrospinal fluid; And it may be a stem cell administration formulation comprising the stem cells suspended in the cerebrospinal fluid.

As used herein, the term "cerebrospinal fluid" may mean a body fluid present in the brain or spinal cord. Specifically, it may be a fluid that is generated in the brain and circulates through the brain and spinal cord along the ventricle and subarachnoid space. It is produced in the choroidal plexus of the brain and is decomposed and absorbed in the arachnoid granules in the subarachnoid space, so that a constant amount is always maintained. Cerebrospinal fluid circulates around the brain and spinal cord, buffering against external shocks, and transporting substances such as hormones and waste products. In one embodiment, the cerebrospinal fluid may be a patient-derived cerebrospinal fluid. In detail, it may be cerebrospinal fluid derived from an individual to be administered stem cells.

In the present specification, stem cells may exist in a suspended state in cerebrospinal fluid. In the present specification, "floating" or "rich" may mean a state in which cells (eg, stem cells) are not attached to the container for culture, but stored in the container for final administration to a patient. More specifically, it may not be stem cells that exist in a suspended state in a culture vessel for 3D culture. Thus, the stem cells may be cells for which QC has been completed within the GMP process. That is, stem cells are cultivated in a culture medium in a GMP facility, and then subjected to a QC (Quality Control) process, and then prepared in a formulation for administration to a patient. At this time, according to the existing technology, the final stem cells for administration to the patient are stored in the same medium as the culture medium.

In one embodiment, the formulation may be patient-specific. Final stem cells for administration to the patient are stored in cerebrospinal fluid (eg, patient-derived). By doing so, the stem cell dosage form maintains stem cell properties, does not change the activity, and can change the expression of some genes or proteins tailored to the patient. Examples of some genes that are tailored to the patient may include TNFα and the like. In the case of the TNFα gene, compared to the case of storage in an existing culture medium (eg, MEMα medium), the expression may be decreased. Therefore, as the stem cells in the formulation according to one embodiment are stored in the cerebrospinal fluid, the expression of the genes or proteins of the stem cells is changed or further activated (e.g., cell regeneration ability, cell mobility) while maintaining stem cell properties. , Activation of genes or proteins related to cell adhesion, angiogenesis, and neuronal regeneration).

In the present specification, the term "stem cell" may refer to an undifferentiated cell having the ability to differentiate into other cells. The stem cells may be embryonic stem cells, adult stem cells, induced pluripotent stem cells, or mesenchymal stem cells. The mesenchymal stem cells may be isolated from humans of various tissues, races, or ages. For example, the mesenchymal stem cells may be adipose tissue-derived, placenta-derived, cord blood-derived, muscle tissue-derived, corneal tissue-derived, or bone marrow tissue-derived mesenchymal stem cells. In addition, for example, the mesenchymal stem cells may be fat stem cells, bone marrow stem cells, cord blood stem cells, neural stem cells, placental stem cells, or cord blood stem cells.

In one embodiment, the formulation may contain a therapeutically effective amount of stem cells. As used herein, a "treatment-effective amount" refers to a patient or individual in need of stem cell therapy, improving the patient's condition (eg, one or more symptoms), delaying the progression of the disease. It means an amount sufficient to produce the desired effect, including, etc. As used herein, "treat" refers to an individual suffering from or at risk of developing a disease, improving the condition of the individual (eg, one or more symptoms), delaying the progression of the disease, symptoms It refers to any form of treatment or prevention that provides an effect, including delaying occurrence or slowing the progression of symptoms. Thus, the term “treatment” also includes prophylactic treatment of an individual that prevents the occurrence of symptoms. The therapeutically effective amount may be, for example, 1.0 x 10 ⁵ to 1.0 x 10 ⁸ cells/kg (body weight), or 1.0 x 10 ⁷ to 1.0 x 10 ⁸ cells/kg (body weight). However, the dosage may be variously prescribed according to factors such as formulation method, administration mode, patient's age, weight, sex, pathological condition, food, administration time, route of administration, excretion rate, and response sensitivity. Taking these factors into account, the dosage can be appropriately adjusted. The number of times of administration can be once or two or more times within the range of clinically acceptable side effects, and the administration site may be administered at one or two or more sites. For non-human animals, the same dose per kg as human or, for example, the volume ratio (e.g., average value) of the target animal and the human organ (heart, etc.) One amount can be administered. Examples of animals to be treated according to an embodiment include humans and mammals for other purposes. Specifically, humans, monkeys, mice, rats, rabbits, sheep, cows, dogs, horses, pigs, etc. Included.

The formulation according to an embodiment may include stem cells and a pharmaceutically acceptable carrier and/or additive as an active ingredient. For example, sterile water, physiological saline, conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives, etc. can do. For topical administration, it may be possible to combine organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymer or copolymer, polyethylene glycol polymer or copolymer, and chemical derivatives thereof. have. When the formulation according to an embodiment is formulated into a formulation suitable for injection, stem cells may be dissolved in a pharmaceutically acceptable carrier or frozen in a dissolved solution state.

In one embodiment, the formulation may include stem cells of 1x10 ⁵ cells to 1 x 10 ⁸ cells or 1x10 ⁵ cells to 1 x 10 ⁷ cells per 0.5 to 10 ml of cerebrospinal fluid.

In one embodiment, the formulation may be for treatment of a disease.

In one embodiment, the present specification provides a method of treating a disease comprising administering to a subject a formulation comprising cerebrospinal fluid and a therapeutically effective amount of stem cells.

Stem cells according to one embodiment, for example, mesenchymal stem cells, may have anti-inflammatory, vascular regeneration, and nerve regeneration effects. Accordingly, the formulation may be usefully used in cell therapy for the prevention or treatment of various diseases including inflammatory diseases, ischemic diseases, and/or neurological diseases. Examples of the ischemic disease include ischemic stroke, myocardial infarction, ischemic heart disease, ischemic brain disease, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, or ischemic leg disease I can. In the present specification, "ischemic stroke" or "stroke" may mean a disease that occurs due to necrosis of brain tissue or cells due to a decrease in cerebral blood flow lasting for a certain time or longer, and "cerebral infarction )" can be used interchangeably. Examples of the nervous system disease may include neurodegenerative diseases. Examples of the neurodegenerative diseases include spinal cord injury, multiple sclerosis, Alzheimer's disease, Frontotemporal dementia, Progressive supranuclear palsy, corticobasal degeneration, and Pick's disease. ), or boxer dementia (Dementia pugilistica, DP).

In one embodiment, the formulation may be administered to a lesion of an individual to obtain a desired effect. For example, the formulation may be a formulation for administration in the brain.

Another aspect provides a method of preparing a dosage form for stem cells.

The method may include maintaining the stem cells to be suspended in the cerebrospinal fluid. The maintaining step may be performed in vitro.

In addition, the stem cell may further include a step of activating gene or protein expression or changing the gene or protein expression of the stem cell to be patient-specific while maintaining stem cellity. Changes in the gene or protein are as described above. Therefore, the method for preparing the dosage form for stem cells is a method for preparing a dosage form for patient-specific stem cells, or a gene or protein (e.g., cell regeneration ability, cell mobility, cell adhesion ability, neovascular function, or nerve regeneration It may be a method for preparing a dosage form of a stem cell with increased expression or activity of a gene or protein associated with the function.

In the method, the maintaining step may be at least 6 hours or longer. The retention time may be a time for which the stem cells are stabilized or for a patient-specific change, for example, 6 hours to a week, or 6 hours to 3 days.

In addition, the maintenance may be to store the stem cells at room temperature. The stem cells may be freeze-dried.

According to the stem cell administration formulation and its manufacturing method according to an aspect, not only is it safe in terms of immunity, but also the expression of the gene or protein of the stem cell is changed or the expression of the gene or protein is further tailored to the patient while maintaining the stem cell property. It has an activated effect.

1A is a view confirming the viability of cells by flow cytometry when umbilical cord-derived mesenchymal stem cells are maintained in MEM medium and cerebrospinal fluid derived from Alzheimer's disease patients according to an embodiment.
1B is a graph quantifying the results of confirming the viability of cells by flow cytometry when umbilical cord-derived mesenchymal stem cells are maintained in MEM medium and cerebrospinal fluid derived from Alzheimer's disease patients according to an embodiment.
2 is a graph confirming the viability of umbilical cord-derived mesenchymal stem cells according to one embodiment by CCK-8 assay.
3A is a flow cytometric analysis result of a positive marker for confirming stem cell properties of umbilical cord-derived mesenchymal stem cells according to an embodiment.
3B is a flow cytometric analysis result of a negative marker for confirming stem cell properties of umbilical cord-derived mesenchymal stem cells according to an embodiment.
Figure 4a is a micrograph confirming the differentiation ability of umbilical cord-derived mesenchymal stem cells according to an embodiment.
Figure 4b is a graph confirming the differentiation ability of umbilical cord-derived mesenchymal stem cells according to an embodiment.
5 is a view showing a change in the RNA expression level of umbilical cord-derived mesenchymal stem cells according to an embodiment.
6 is a view showing a change in the RNA expression level of umbilical cord-derived mesenchymal stem cells according to an embodiment; Green indicates a decrease in expression level, black indicates no change in expression level, and red indicates an increase in expression level.
7 is a diagram showing changes in the RNA expression level of umbilical cord-derived mesenchymal stem cells according to one embodiment.
8A is a result of comparing the gene expression level of umbilical cord-derived mesenchymal stem cells stored in patient-derived cerebrospinal fluid with umbilical cord-derived mesenchymal stem cells stored in cerebrospinal fluid derived from a normal person according to an embodiment; Red: increased gene expression; Green: decreased gene expression.
8B is a diagram showing genes increased in umbilical cord-derived mesenchymal stem cells stored in patient-derived cerebrospinal fluid compared to umbilical cord-derived mesenchymal stem cells stored in cerebrospinal fluid derived from a normal person according to an embodiment.
8C is a view showing a table comprehensively analyzing the functions of genes increased in umbilical cord-derived mesenchymal stem cells stored in patient-derived cerebrospinal fluid according to an embodiment.
9 is a diagram showing the results of analyzing the functions of the genes increased in FIG. 8C; a: cellular component; b: molecular function.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1. Isolation of mesenchymal stem cells

Mesenchymal stem cells were used as umbilical cord-derived mesenchymal stem cells.

Specifically, informed consent was obtained in advance from a healthy mother who delivered normally, and the umbilical cord of about 15 cm to about 20 cm was separated at the time of the first cesarean section. The separated umbilical cord was washed 2 to 5 times with PBS to remove blood. Thereafter, the umbilical cord was cut to a size of about 1.5 cm, and in order to separate the cells from the Wharton jelly, each piece was removed from blood in the blood vessel using forceps. After each piece was cut with sterilized scissors, the cord blood was removed. After the gelatin pieces surrounding the blood were cut into a size of 1 to 3 mm, collagenase I was added and reacted in a shaking incubator. After filtering the degraded tissue using a strainer, 10% FBS (Biowest, Nuaille', France), 0.5% gentamicin (10 mg/ml) (Gibco, NY, USA), and aMEM (a- modified Minimum Essential Media) (Gibco, NY, USA). Culture conditions were maintained at 37°C and 5% CO ₂ conditions. Thereafter, the culture medium was changed every 3 to 4 days to remove cells that did not adhere to the bottom of the flask. The first passage was performed by reacting the cells attached to the bottom of the flask with TrypLE, an animal component free (ACF) recombinant enzyme, in an incubator at 37° C. for about 3 minutes to recover mesenchymal stem cells.

Experimental Example 1. Analysis of maintenance of viability of mesenchymal stem cells

The umbilical cord-derived mesenchymal stem cells isolated in Example 1 were stored at 5.5 x 10 ⁶ cells in a vial containing MEM medium (1.5 ml), and 5.5 in a vial containing cerebrospinal fluid derived from Alzheimer's disease patient (1.5 ml). Stored as x 10 ⁶ cells, confirmed through flow cytometry. The flow cytometry results are shown in FIG. 1. In all figures, CSF1 to CSF4 are identification numbers of cerebrospinal fluid derived from Alzheimer's disease patients.

In addition, the viability of cells was analyzed through the CCK-8 assay. Specifically, mesenchymal stem cells suspended and maintained for each formulation (MEM medium, and cerebrospinal fluid) were dispensed into a 96-well plate at 3 x 10 ³ cells per well, and survival was analyzed for 72 hours in units of 24 hours. After cell adhesion was confirmed, a CCK-8 solution (10ul) was added to each well, and incubated at 37°C for 1 hour. The absorbance of CCK-8 was analyzed at 450 nm with a microplate reader (x-Mark ^™ , Bio-Rad Laboratories, Inc), and the results are shown in FIG. 2.

1A is a view confirming the viability of cells by flow cytometry when umbilical cord-derived mesenchymal stem cells are maintained in MEM medium and cerebrospinal fluid derived from Alzheimer's disease patients according to an embodiment.

1B is a graph quantifying the results of confirming the viability of cells by flow cytometry when umbilical cord-derived mesenchymal stem cells are maintained in MEM medium and cerebrospinal fluid derived from Alzheimer's disease patients according to an embodiment.

2 is a graph confirming the viability of umbilical cord-derived mesenchymal stem cells according to one embodiment by CCK-8 assay.

As shown in Figs. 1 and 2, it can be seen that even if the umbilical cord-derived mesenchymal stem cells are maintained in the patient-derived cerebrospinal fluid, there is no difference in viability of the cells compared to the control group.

Experimental Example 2. Analysis of maintenance of stem cell properties of mesenchymal stem cells

Mesenchymal stem cells were maintained in MEM medium and patient-derived cerebrospinal fluid in the same manner as in Experimental Example 1. The stem cell properties of the cells were analyzed.

As positive surface markers for stem cell ability, CD90, CD73, CD105, and CD166 were used, and as negative surface markers, CD14, CD11b, CD19, CD34, CD45, and HLA-DR were used. Specifically, for flow cytometry, cells were washed using DPBS, and then placed in DPBS containing 2% FBS and CD90, CD73, CD105, CD166, CD14, CD11b, CD19, CD34, CD45, and HLA-DR markers on ice. It was allowed to react for 20 minutes. Thereafter, the surface antigen was analyzed through flow cytometry (FACS Calibur, Becton Bickinson), and the results are shown in FIG. 3.

In addition, the differentiation ability of stem cells was analyzed as follows.

For the analysis of adipocyte differentiation ability, umbilical cord-derived mesenchymal stem cells were placed in adipogenesis differentiation media (StemPro® Adipogenesis Differentiation Kit, Life Technology) and cultured while changing the medium every 3 days for 2 weeks. Thereafter, the culture solution was removed, washed with Ca/Mg free DPBS, and then 4% paraformaldehyde was added and reacted at room temperature for 15 minutes. After washing with 60% isopropanol, Oil Red O was added and reacted for 10 minutes, and after washing with purified water, adipocytes were observed under a microscope, and the results are shown in FIG. 4.

For the analysis of bone cell differentiation ability, umbilical cord-derived mesenchymal stem cells were placed in a bone formation differentiation media (StemPro® Osteogenesis Differentiation Kit, Life Technology) and cultured while changing the media every 3 days for 2 weeks. After that, the culture solution was prepared, washed with Ca/Mg free DPBS, and then 4% paraformaldehyde was added and reacted at room temperature for 15 minutes. After the reaction is complete, wash with purified water, add 1% silver nitrate solution, react at room temperature for 5 minutes, wash with purified water, add 5% sodium thiosulfate solution, and then at room temperature. It was reacted for 5 minutes. Next, after washing with purified water, 0.1% Nuclear Fast Red Solution was added and reacted at room temperature for 5 minutes. Thereafter, the samples were washed with purified water and the calcium accumulated samples were analyzed under a microscope, and the results are shown in FIG. 4.

For the analysis of chondrocyte differentiation ability, umbilical cord-derived mesenchymal stem cells were placed in chondrogenesis differentiation media (StemPro® Chondrogenesis Differentiation Kit, Life Technology), and the medium was removed every 3 days for 3 weeks with the lid loosely closed. It was cultured while exchanging. The cell mass was made into a paraffin block, cut, and stained with Alcian blue. Thereafter, the blue-stained chondrocytes were analyzed with an optical microscope, and the results are shown in Fig. 4A and quantified and shown in Fig. 4B.

3A is a flow cytometric analysis result of a positive marker for confirming stem cell properties of umbilical cord-derived mesenchymal stem cells according to an embodiment.

3B is a flow cytometric analysis result of a negative marker for confirming stem cell properties of umbilical cord-derived mesenchymal stem cells according to an embodiment.

Figure 4a is a micrograph confirming the differentiation ability of umbilical cord-derived mesenchymal stem cells according to an embodiment.

Figure 4b is a graph confirming the differentiation ability of umbilical cord-derived mesenchymal stem cells according to an embodiment.

As shown in Figure 3, compared to the umbilical cord mesenchymal stem cells stored in the culture medium MEM-a as a conventional formulation, the marker expression of the umbilical cord mesenchymal stem cells stored in the cerebrospinal fluid (AD CSF) derived from Alzheimer's disease patients is significantly changed. It was confirmed that it did not.

In addition, as shown in FIG. 4, it was confirmed that the umbilical cord-derived mesenchymal stem cells stored in cerebrospinal fluid (AD CSF) maintained the ability to differentiate into adipocytes, bone cells, and cartilage cells.

Experimental Example 3. Analysis of gene expression level of mesenchymal stem cells

RNA was extracted from stem cells to analyze genes expressed in umbilical cord-derived mesenchymal stem cells, followed by labeling and purification. The labeled cDNA was hybridized to an Illumina Expression BeadChip and the result was derived. After statistical processing of the data, the results are shown in FIGS. 5 to 7.

The confirmed RNA expression level analysis list is as follows.

As shown in Fig. 5, Stemness Markers: FGF2, INS, LIF, POU5F1, SOX2, TERT, WNT3A, ZFP42; MSC-Specific Markers: ALCAM, ANPEP, BMP2, CASP3, CD44, ENG, ERBB2, FUT4, FZD9, ITGA6, ITGAV, KDR, MCAM, NGFR, NT5E, PDGFRB, PROM1, THY1, VCAM1; Other Genes Associated with MSC: ANXA5, BDNF, BGLAP, BMP7, COL1A1, CSF2, CSF3, CTNNB1, EGF, FUT1, GTF3A, HGF, ICAM1, IFNG, IGF1, IL10, IL1B, IL6, ITGB1, KITLG, MITF, MMP2, NES, NUDT6, PIGS, PTPRC, SLC17A5, TGFB3, TNF, VEGFA, VIM, VWF; MSC Differentiation Markers; (i) Genes Involved in Osteogenesis: BMP2, BMP6, FGF10, HDAC1, HNF1A, KDR, PTK2, RUNX2, SMURF1, SMURF2, TBX5; (ii) Gene Involved in Adipogenesis: PPARG, RHOA, RUNX2; (iii) Gene Involved in Chondrogenesis: ABCB1, BMP2, BMP4, BMP6, GDF5, GDF6, GDF7, HAT1, ITGAX, KAT2B, SOX9, TGFB1; (iv) Gene Involved in Myogenesis: JAG1, NOTCH1; (v) Gene Involved in Tenogenesis (Tendon Development): BMP2, GDF15, SMAD4, and TGFB1 are related to stem cell properties, and it was found that there was no significant change depending on the patient.

However, as shown in FIGS. 6 and 7, it was found that some genes, for example, TNFα, showed differences in expression patterns for each patient. In particular, a decrease in inflammation-related factors in common was confirmed.

As a result of the above, when stored in patient-derived cerebrospinal fluid, it was confirmed that the gene expression of umbilical cord-derived mesenchymal stem cells changed patiently.

In addition, when umbilical cord mesenchymal stem cells were exposed to cerebrospinal fluid of a patient with Alzheimer's disease and when exposed to normal cerebrospinal fluid, changes in RNA expression levels of cells were compared.

Specifically, mesenchymal stem cells were stored in the same manner as in Example 1, except that normal human-derived cerebrospinal fluid was used. Using the MeV program, gene expression of mesenchymal stem cells was analyzed in the same manner as in the above experiment, and the results are shown in FIG. 8.

In addition, as a result of the gene expression analysis, Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to analyze what functions each expressed gene had, and the results are shown in FIG. 9. The fold enrichment and count in FIG. 9 indicate how many genes are related to the function shown in the results. Count indicates how many genes are involved in the function. Fold enrichment is calculated as the ratio of the genes involved in the function among the total genes that were analyzed / the ratio of the genes involved in the function in the known human genome.

8A is a result of comparing the gene expression level of umbilical cord-derived mesenchymal stem cells stored in patient-derived cerebrospinal fluid with umbilical cord-derived mesenchymal stem cells stored in cerebrospinal fluid derived from a normal person according to an embodiment; Red: increased gene expression; Green: decreased gene expression.

8B is a diagram showing genes increased in umbilical cord-derived mesenchymal stem cells stored in patient-derived cerebrospinal fluid compared to umbilical cord-derived mesenchymal stem cells stored in cerebrospinal fluid derived from a normal person according to an embodiment.

8C is a view showing a table comprehensively analyzing the functions of genes increased in umbilical cord-derived mesenchymal stem cells stored in patient-derived cerebrospinal fluid according to an embodiment.

9 is a diagram showing the results of analyzing the functions of the genes increased in FIG. 8C.

As shown in Figure 8, both umbilical cord mesenchymal stem cells exposed to cerebrospinal fluid derived from a normal person and umbilical cord mesenchymal stem cells exposed to patient-derived cerebrospinal fluid are activated, but the gene of umbilical cord mesenchymal stem cells exposed to patient-derived cerebrospinal fluid is normal It was confirmed that the umbilical cord mesenchymal stem cells exposed to cerebrospinal fluid were slightly more active. In addition, although there are many genes that are commonly activated in patient-derived cerebrospinal fluid, there are also genes that are activated in each, indicating that there is also a patient-specific gene activation response. Subsequently, as a result of analyzing the functions of genes commonly increased in mesenchymal stem cells exposed to patient-derived cerebrospinal fluid, it was confirmed that functions by secreted factors such as signal peptide, growth factor activity, or cytokine activity were greatly increased. In addition, it was confirmed that the functions of activating cell mobility and regeneration ability such as neovascularization, cell proliferation, cell migration, cell adhesion, and neural formation were greatly increased.

As shown in FIG. 9, as a result of confirming where the cellular components are located in the cell, it was confirmed that most of the expression-increasing genes were expressed or secreted in the cell membrane and out of the cell. In addition, it was found that molecular functions are related to various growth factor activities, cell adhesion, and cytokine activity. The above result means that the mesenchymal stem cells stored in the cerebrospinal fluid according to an embodiment can be used as a cell therapy agent by secreting several active proteins.

Claims (12)

A patient-specific dosage form of stem cells for the treatment of neurodegenerative diseases, comprising autologous cerebrospinal fluid of a neurodegenerative disease person and a therapeutically effective amount of human umbilical cord-derived mesenchymal stem cells suspended in the cerebrospinal fluid. delete delete The dosage form according to claim 1, wherein the administration is intracranial administration. The dosage form according to claim 1, wherein the neurodegenerative disease is Alzheimer's disease. The dosage form according to claim 1, comprising 1x10 ⁵ to 1x10 ⁸ stem cells per 0.5 to 10 ml of cerebrospinal fluid. The dosage form according to claim 1, wherein as the stem cells are stored in the cerebrospinal fluid, the expression of genes or proteins of the stem cells is changed or further activated in a patient-specific manner while maintaining stem cell properties. Cell therapy for the treatment of neurodegenerative diseases, comprising the formulation of any one of claims 1, 4 to 7. In vitro human umbilical cord-derived mesenchymal stem cells of a patient-specific stem cell for the treatment of neurodegenerative diseases comprising the step of maintaining 1x10 ⁵ to 1 x 10 ⁸ per 0.5 to 10 ml of autologous cerebrospinal fluid of a neurodegenerative disease Method of making dosage form. The method according to claim 9, wherein the stem cell further comprises the step of further activating the gene or protein expression of the stem cell while maintaining the stem cellity and changing the gene or protein expression of the stem cell to be patient-specific. The method of claim 9, wherein the maintaining step is at least 6 hours or longer. The method of claim 9, wherein the neurodegenerative disease is Alzheimer's disease.

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