KR100823090B1 - Composition for cell therapy of spinal cord injury with stem cell derived from umbilical cord blood - Google Patents

Abstract

The present invention relates to a cell therapy method for treating a paraspinal paraplegia patient by completely exposing a patient's spinal cord using umbilical cord blood-derived stem cells to transplant the stem cells. Since it is possible to solve the immune rejection reaction without an immunosuppressive agent, there is no possibility of teratoma or terato carcinoma in the human body like other embryonic stem cells, the cell therapy according to the present invention is spinal cord It is expected to bring new hope to paralyzed patients.

Spinal paraplegia, cord blood stem cells, immunosuppressive agents, immune rejection, histocompatibility genes, motor nerves, sensory nerves, neuronal differentiation.

Description

COMPOSITION FOR CELL THERAPY OF SPINAL CORD INJURY WITH STEM CELL DERIVED FROM UMBILICAL CORD BLOOD}

Figure 1 shows the cell properties of umbilical cord blood-derived stem cells show CD13 positive, CD29 positive, SH2 positive, ASMA positive.

Figure 2 shows the expression of neuronal genes after culturing cord blood-derived stem cells in neuronal cell culture.

The present invention relates to a cell therapy method for treating a spinal paraplegia patient by transplanting stem cells by completely exposing the spinal cord of the patient using stem cells derived from umbilical cord blood-derived stem cells matching the patient and histocompatibility genes. do.

In the medical field, many advances have been made, but there are diseases that cannot be cured by modern medicine, that is, incurable diseases. In the condition of patients with these intractable diseases, tissues and cells that should function as a whole often lose their function partially or entirely. In this case, it is true that fundamental treatment is impossible with any known drugs or surgical treatments.

Patients with paraplegia after spinal cord injury are included in intractable disease. Spinal cord injury is a disease or accident that causes damage to the spinal cord and affects the performance of sensory and motor signals that will pass through the damaged area, resulting in sensory or motor neurons. It means that the communication will be interrupted. Spinal cord injuries caused by such diseases or accidents cause impaired physical function due to improper transmission of motor or sensory nerves between the brain and the body and are called a disabled person with the spinal cord injury. Spinal cord injury mainly measures the degree of paralysis according to Frankel's scale. The degree of disability varies depending on the area of injury of the spinal cord and the degree of damage, including complete limb paralysis and partial paralysis of the lower extremities.

A serious problem for society with spinal cord injuries is that people with active age are less than 40 years old (about 80 percent). In addition, most people with spinal cord disorders are acquired disorders and suffer a lot physically and mentally due to sudden change of environment and various limitations of social life. Currently, medical care focuses on care to prevent other complications (respiratory care, gastrointestinal therapy, pressure sores, prevention of thrombosis) rather than active treatment for paralyzed patients with spinal cord disorders. .

Traditionally, when organ function is partially paralyzed or completely lost, the last option is to transplant the organ. In this case, it is difficult to find an organ suitable for the patient, and there is a problem that a lifetime immunosuppressant should be used even if the patient is found and transplanted into the patient. Immunosuppressants not only reduce the quality of life of the patient, but also lead to a decrease in immunity, resulting in a number of complications. Moreover, spinal palsy suffers from the fact that unlike other incurable diseases, there is no way to transplant certain tissues or organs.

On the other hand, stem cells (i.e. stem cells) are immature cells whose fate has not yet been determined. When the organs or tissues in the body are shocked or damaged from the outside, they refer to cells that have the ability to differentiate themselves. Depending on the source, it is divided into embryonic stem cells, adult stem cells, and neonatal stem cells. Currently, it is easy to isolate and culture stem cells from bone marrow, but it is not easy to obtain bone marrow, and it is known that it is practically difficult to solve the immune rejection reaction when transplanting stem cells of another person. In contrast, cord blood is easier to acquire than bone marrow, and if you have a large number of cord blood units, you can use a cord blood stem cell that matches or matches the histocompatibility gene of the patient. This has the advantage of being solved.

An object of the present invention is to construct a novel cell therapy for patients with spinal paraplegia who cannot be treated in modern medical conditions using cord blood-derived stem cells with matching genes, and the cord blood-derived stem cells provided to patients can be rapidly transferred to neurons in vitro. By confirming their differentiation, they are trying to provide new therapies by inducing spinal palsy patients who can't radically regenerate their nerves.

In the present invention to achieve the above object,

Pure umbilical cord blood within 24 hours of birth, adding anticoagulant to umbilical cord blood with a volume of at least 45 ml per unit;

Umbilical cord blood mixed with anticoagulant was diluted with alpha-minimum essential medium (aMEM) medium and centrifuged to harvest monocytes; And,

The obtained monocytes include Stem Cell Factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3) and interleukin-6 (IL-6). Comprising umbilical cord blood-derived mesenchymal stem cells obtained by suspension culture in aMEM medium,

Provided are compositions for implantation in a spinal cord of a patient for treatment of a paraspinal paraplegia patient.

In the present invention,

Thaw frozen cord blood to dilute with alpha-minimum essential medium (aMEM) medium and centrifuge to harvest monocytes;

CD133 positive cells were isolated from the obtained monocytes; And,

Stem Cell Factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3) and interleukin-6 (IL-6) Umbilical cord blood-derived mesenchymal stem cells obtained by suspension culture in the aMEM medium included,

Provided are compositions for implantation in a spinal cord of a patient for treatment of a paraspinal paraplegia patient.

In order to achieve the above another object, in the present invention

Selecting cord blood with 6 HLA genes or 1 or 2 discrepancies with the patient;

Isolating and culturing stem cells from said selected cord blood; And,

It provides a method of treating spinal paraplegia, comprising the step of transplanting the cultured stem cells into the spinal cord.

In the present invention, using a cord blood stem cell with a matched histocompatibility gene and a spinal paraplegic patient, which cannot be treated by modern medicine, constructing a new cell therapy method for transplanting stem cells by exposing the spinal cord of the patient completely, and in vitro Culture combinations were used to induce and confirm rapid differentiation of cord blood derived stem cells into neurons.

In contrast to conventional stem cell treatment, the stem cells are not directly injected into the damaged area, but are injected into the blood vessel (vein). In the present invention, stem cells are directly injected into the damaged area to deliver the maximum effect. A new method is adopted.

Considering the fact that there is no active treatment for paralysis patients due to spinal cord disorder, and transplantation is impossible, cell therapy of patients with spinal paraplegia using cord blood-derived stem cells according to the present invention is a valuable report that has no origin worldwide. . To date, no stem cells have been treated with spinal palsy patients worldwide. However, there was an example of using an animal.

Spinal cord complete palsy cell therapy using umbilical cord blood-derived stem cells according to the present invention is based on the match between the tissue compatible genes of the patient and the stem cells, immunosuppression by preparing a cord blood stem cells matching the tissue compatible genes in advance for the patient The reaction can be solved. Accordingly, there is no need to use an immunosuppressive agent in the injection of cells for regenerating the nerves of the patient. Therefore, there is a significance in improving not only the treatment of the patient but also the future quality of life.

In addition, in the present invention, the cord blood-derived stem cells were treated with a newly combined reagent in vitro to confirm rapid differentiation into neurons. In other words, when differentiating into neural cells from conventional bone marrow stem cells, differentiation into neural cells occurs depending on cAMP. The cord blood-derived stem cells according to the present invention are not cAMP, 8-bromo-cAMP, or forskolin. DMSO and antioxidants, such as antioxidants, could differentiate cord blood-derived stem cells into neurons in 24 hours. Through this method, it was confirmed that the umbilical cord blood-derived stem cells according to the present invention are differentiated into glia cells, which are early stages of nerve development, and other neurons.

Moreover, since cord blood stem cells are unlikely to develop teratoma or terato carcinoma in the human body like other embryonic stem cells, the cell therapy according to the present invention is expected to bring new hope to patients with spinal palsy. do.                     

Hereinafter, the cell treatment method of paraspinal paraplegia using cord blood-derived stem cells according to the present invention will be described in detail through Examples. However, these Examples are only illustrative of the present invention, and the scope of the present invention is not limited to these.

Example 1 Screening of Umbilical Cord Blood

After determination of the six histocompatibility antigen (HLA) genes matched with the patient, the frozen stored cord blood with the same or one or two mismatched HLA genes were selected.

Histocompatibility antigens are an important factor in determining whether or not cells are engrafted when foreign cells are injected into the body. Histocompatibility antigens are based on DNA for testing all six genes, and the six genes are based on the match of class I A, B and class II DR.

Example 2-1: Isolation and Culture of Stem Cells from Umbilical Cord Blood

To separate monocytes from umbilical cord blood, dilute umbilical cord blood with a double dose of aMEM (alpha-minimum essential medium, Jeil Biotech Services, Korea), transfer to 50 ml falcon tubes, and centrifuge at 300xg for 10 minutes at room temperature. It was. The separated buffy coat layer was harvested and diluted again with a double volume of aMEM, then superimposed on Ficoll-Hypaque and centrifuged at 300xg for 30 minutes at room temperature.

Ficoll-Hypaque, a polymer of Ficoll (a polymer of sucrose) and Hypaque (sodium ditrizoate), is mainly used to separate monocytes from blood. Ficoll-Hypaque specific gravity is 1.077 g / ㎖, monocytes are lighter than this, but red blood cells are heavier than this can be separated by specific gravity. In other words, when blood is centrifuged on Ficoll-Hypaque, monocytes are collected on Ficoll-Hypaque.

The monocytes obtained by such a density gradient centrifugation method were placed in aMEM for washing with no additives and centrifuged at 200 × g for 10 minutes, and then washed by discarding aMEM except for the cells that settled on the bottom of the falcon tube. One more time aMEM was added and centrifuged at 200 × g for 10 minutes, after which the aMEM was discarded except for the cells that settled on the bottom of the Falcon tube.

Next, antibiotics (1000 U / ml penicillin G, 1000 μg / ml streptomycin sulfate, Gibco-BRL), antifungal agents (0.25 μg / ml amphotericin B), and 2 mM glutamine (Slutma) were included. Stem Cell Factor (50 ng / ml), GM-CSF (granulocyte-macrophage colony-stimulating factor; 10 ng / ml as cell growth factor with 20% fetal bovine serum (FBS; fetal bovine serum, Jeil Biotech Services) in aMEM medium ), G-CSF (granulocyte colony-stimulating factor (10 ng / ml), IL-3 (interleukin-3; 10 ng / ml) and IL-6 (interleukin-6; 10 ng / ml) It was suspended at a concentration of 1 × 10 ⁶ / cm 2.

The floating cells were removed from the cell populations cultured for 5 days, and after the adherent cells were secured, the cells were incubated for 25 days by completely replacing the cells with aMEM containing 20% fetal calf serum and antibiotics every two days without washing.

Example 2-2 Isolation and Culture of Stem Cells from Cryopreserved Cord Blood

Umbilical cord blood, which was stored frozen at minus 196 ° C, was thawed immediately in a 37 ° C water bath. In order to separate monocytes from umbilical cord blood, umbilical cord blood was diluted with a double dose with aMEM (alpha-minimum essential medium, Jeil Biotech Services, Korea) and centrifuged at 300xg for 10 minutes at room temperature. The separated buffy coat layer was harvested and diluted again with a double volume of aMEM, superimposed on Ficoll-Hypaque, and centrifuged at 300xg for 30 minutes at room temperature.

Ficoll-Hypaque, a polymer of Ficoll (a polymer of sucrose) and Hypaque (sodium ditrizoate), is mainly used to separate monocytes from blood. Ficoll-Hypaque has a specific gravity of 1.007 g / mL, and monocytes are lighter than this but erythrocytes are heavier than that, so they can be separated by specific gravity. In other words, when the blood is centrifuged on Ficoll-Hypaque, monocytes are collected on Ficoll-Hypaque.

The monocytes obtained by such a density gradient centrifugation method were washed twice with aMEM for washing with no additives.

The obtained monocytes were positively isolated by CD133 isolation kit (Isolation kit: Miltenyi Bioteck, Germany). The separation method was first added 100 μl of blocking reagent to the obtained monocytes to remove non-specific binding, and then 100 μl of CD133 / Microbead was mixed well to make 500 μl of the total volume and 30 ° C. at 4 ° C. Incubate for minutes. After centrifugation (300xg, 10 minutes) by adding 10-fold PBS (D-phosphate buffered saline, Jeil Biotech Services, Korea), discard the PBS except the cells attached to the tube and resuspend in 500 μl PBS. (resuspension). The column was then washed with 3 ml PBS buffer beforehand, and the resuspended cells were placed in a column attached to the machine for at least 15 minutes and then rinsed four times with PBS. Next, the column was removed from the machine, placed in a tube and flushed with a plunger with PBS appropriately selected to select positive cells.

Selected cells included antibiotics (1000 U / ml penicillin G, 1000 μg / ml streptomycin sulfate, Gibco-BRL), antifungal agents (0.25 μg / ml amphotericin B), and 2 mM glutamine (Sigma) Stem Cell Factor (50 ng / ml), GM-CSF (granulocyte-macrophage colony-stimulating factor; 10 ng /) as cell growth factor with 20% fetal bovine serum (FBS; fetal bovine serum, Jeil Biotech Services) Ml), granulocyte colony-stimulating factor (G-CSF) (10 ng / ml), interleukin-3 (10 ng / ml) and IL-6 (interleukin-6; 10 ng / ml), The cells were suspended at a concentration of 1 × 10 ⁶ / cm 2.

The floating cells were removed from the cell populations cultured for 5 days, and after the adherent cells were secured, the cells were incubated for 25 days by completely replacing the cells with aMEM containing 20% fetal calf serum and antibiotics every two days without washing.

Example 3: Confirmation of Differentiation of Cord Blood-derived Stem Cells into Neurons (in vitro)

In Example 2, 0.05% trypsin-EDTA was added to the stem cells cultured for 2 weeks, and the attached cells were suspended. Then, the cells were cultured at 1 × 10 ⁶ cells / cm 2 with 20% low-glucose DMEM. . After culturing the cells for 2 days and discarding the culture medium, the cells attached with low-glucose DMEM were washed. After washing, nerve cells were induced for 24 hours in a medium containing 10 ng bFGF in low-glucose DMEM containing 20% fetal bovine serum. Thereafter, the cells attached with D-PBS, which were previously warmed to 37 ° C., were washed once and 25 mM KCl, 2% dimethylsulfoxide (DMSO), 100 μM butylated hydroxyanisole (BHA), Nerve in low-glucose DMEM culture containing 5 μg / ml insulin, 100 μg / ml transferrin, 20 nM progesterone, 100 μM putrescine, 20 nM sodium selenite and 20 ng / ml bFGF Cell differentiation was induced.

Figure 1 shows the cell properties of umbilical cord blood-derived stem cells show CD13 positive, CD29 positive, SH2 positive, ASMA positive.

Figure 2 shows the expression of neuronal genes after culturing cord blood-derived stem cells in neuronal cell culture. As shown here, the neuronal morphology was shown in 24 hours, and the neuronal genes Tuj1, TrkA, GFAP, and CNPase were expressed.

Clinical Trial: Results of Patients with Paraplegia

1. History of spinal cord paralysis patients

(1) Patient: Hwang OO (Female, 37 years old)

(2) Cause: Falling from Gacheon more than 20 years ago, causing paraspinal paraplegia

(3) disease: paraplegia complete paralysis. Complete paralysis below thoracic spine 9 (left) and thoracic spine 12 (right)

2. Surgical Therapy                     

Using stem cells derived from umbilical cord blood with matching histocompatibility genes, the stem cells were transplanted directly to the thoracic spine 9 with the spinal cord fully exposed for 5 hours from 8 am to 1 pm on October 12. It was.

Specifically, the stem cells were cultured for about 3 weeks in 0.05% trypsin-EDTA was allowed to react at room temperature for 5 minutes, and then D-PBS was centrifuged at 300Xg for 10 minutes. After centrifugation, 5 ml of physiological saline was added to the cells remaining in the test tube, and centrifugation at 300Xg for 10 minutes was repeated three times to wash the stem cells to be as clean as possible without other compositions. Finally, the stem cells remaining in the test tube were adjusted to 5 × 10 ⁷ cells in a total of 6 ml of physiological saline, and the resuspended cells were injected directly into the injured spine directly with a syringe.

After the stem cell transplantation, the exposed spinal cord was sealed again with surgical treatment, and then examined for any neurological changes in the lower body of the patient.

3. Surgical Results

Dermatomal Somatosensory Evoked Potentials (SEP) Somatosensory Evoked Potentials: The somatosensory Evoked Potentials test electrically stimulates the sensory nerves in the upper and lower extremities, and then inserts a recording electrode into the cerebral cortex where the stimulated nerves are housed. This is a test method to record This somatosensory development test stimulates somatosensory of the hands or feet and analyzes the electrical signals generated in the sensory cortex of the brain to improve where and when there are abnormalities from the hands or legs to the sensory cortex of the brain. Can be diagnosed.

The following table shows the Dermatomal SEP results before and after transplantation. Table 1 shows the right side and Table 2 shows the left side. For reference, the spine (backbone) is composed of 31 nodes and bones, from the top to the cervical spine (C1-C9), thoracic vertebrae (T1-T12), lumbar vertebrae (L1-L5), and lumbar vertebrae (S1-S5). Is done.

Spinal cord October 11 October 19 October 26 November 1 T10 31.4 / 42.4 27.8 / 39.2 32.0 / 41.2 30.4 / 38.8 T11 34.2 / 42.2 34.6 / 45.2 40.2 / 47.2 48.8 / 54.8 T12 50.4 / 64.8 51.2 / 61.4 49.2 / 59.6 51.6 / 58.6 L1 no response 51.4 / 60.8 no response 66.0 / 73.4 L2 no response no response no response 74.8 / 81.2 L3 no response no response no response no response L4 no response no response no response no response L5 no response no response no response no response

Spinal cord October 11 October 19 October 26 November 1 T9 29.4 / 43.4 33.8 / 45.2 23.6 / 34.4 35.2 / 45.4 T10 42.2 / 49.6 36.6 / 49.8 36.8 / 42.8 36.8 / 44.4 T11 46.8 / 50.8 37.4 / 48.6 40.8 / 46.4 39.6 / 53.4 T12 no response 44.6 / 64.2 53.8 / 63.6 45.4 / 55.2 L1 no response no response 64.4 / 77.6 58.4 / 70.2 L2 no response no response no response 72.0 / 83.2 L3 no response no response no response no response L4 no response no response no response no response L5 no response no response no response no response

As shown in Tables 1 and 2 above, the results of the dermatomal SEP on the right side after transplantation showed no response at all on the initial October 11 in lumbar spine 1 (L1). It appeared suddenly and finally the nerves were regenerated on November 1st. In addition, the lumbar spine 2 (L2) also can be confirmed that the first responsiveness on November 1 the nerve revived. The left nerve also had no response at all on the thoracic spine 12 (T12) and began to respond on October 19, exactly three days later. On November 1, you can see that the nerves have come down to the lumbar spine twice.

As described above, according to the spinal cord complete palsy cell treatment method using the cord blood-derived stem cells according to the present invention, since the histocompatibility genes of the patient and the stem cells are identical, the immune rejection reaction can be solved without immunosuppressive agents. As with other embryonic stem cells, there is no possibility of teratoma or terato carcinoma occurring in the human body, and the cell therapy according to the present invention is expected to bring new hope to patients with spinal palsy.

Claims (3)

Pure umbilical cord blood within 24 hours of birth, adding anticoagulant to umbilical cord blood with a volume of at least 45 ml per unit; Umbilical cord blood mixed with anticoagulant was diluted with alpha-minimum essential medium (αMEM) medium and centrifuged to harvest monocytes; And, The obtained monocytes include Stem Cell Factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3) and interleukin-6 (IL-6). Comprising umbilical cord blood-derived mesenchymal stem cells obtained by suspension culture in the prepared αMEM medium, A composition for implantation in a spinal cord of a patient for treatment of a paraspinal paraplegia patient. Thaw frozen cord blood, dilute with alpha-minimum essential medium and centrifuge to harvest monocytes; CD133 positive cells were isolated from the obtained monocytes; And, Stem Cell Factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3) and interleukin-6 (IL-6) Umbilical cord blood-derived mesenchymal stem cells obtained by suspension culture in the contained αMEM medium, A composition for implantation in a spinal cord of a patient for treatment of a paraspinal paraplegia patient. delete

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