WO2006054883A1

WO2006054883A1 - Composition for cell therapy of spinal cord injury with stem cell derived from umbilical cord blood

Info

Publication number: WO2006054883A1
Application number: PCT/KR2005/003949
Authority: WO
Inventors: Hoon Han
Original assignee: Hoon Han
Priority date: 2004-11-22
Filing date: 2005-11-22
Publication date: 2006-05-26
Also published as: KR20060056615A; KR100823090B1

Abstract

Provided is a cell therapy for treating a patient with spinal paraplegia, comprising transplanting stem cells into a spinal cord of a patient under complete exposure of the spinal cord, using an umbilical cord blood-derived stem cell. The cell therapy in accordance with the present invention is based on the condition that the histocompatibility antigens of a stem cell are identical with those of a patient and thus can solve the problems associated with immune rejection without use of immunosuppressant drugs. Further, it is advantageous in that unlike the cases in which any other embryonic stem cells are employed, the cell therapy of the present invention presents no possibility of teratoma or teratocarcinoma occurrence in the body, and thus it is expected that the cell therapy in accordance with the present invention will give promising hope of a possible treatment for spinal paraplegic patients.

Description

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

Technical Field

The present invention relates to a cell therapy for treating a patient with spinal paraplegia, comprising transplanting a stem cell into a spinal cord of the patient under complete exposure of the spinal cord, using an umbilical cord blood- derived stem cell which is based on a stem cell having histocompatibility antigens that are identical with those of patients.

Background Art

Despite remarkable advancements in a medical field owing to developments in science and technology, there still remain a variety of intractable diseases which are beyond current medical treatments. In many cases, people suffering from such incurable diseases generally manifest conditions accompanied by partial or complete loss of normal functions that should be performed by the corresponding tissues and cells. In fact, it seems impossible to achieve fundamental treatment of such conditions by any drug or surgical therapy that is currently known in the related art.

People suffering from spinal paraplegia after spinal cord injury can also be defined as patients with intractable diseases. Spinal cord injury refers to conditions in which diseases or accidents result in injury of the spinal cord, thus in turn affecting performance of sensory signals and motor signals which will pass through injured sites, and as a result, leading to occurrence of disorder and dysfunction in transmission of sensory neurons or motor neurons. A disabled person with the spinal cord injury refers to a person who suffers from disorders in physical functions due to failure to normally and sufficiently transmit motor neurons or sensory neurons between the cerebrum and body, resulting from spinal cord injury caused by such diseases or accidents. Extent and severity of spinal cord injury is determined by measuring a degree of paraplegia according to Frankel Grade. Spinal paraplegia may include various forms such as complete quadriplegia, partial paraplegia of lower extremities and the like, depending upon impaired sites of the spinal cord and the extent and severity of spinal cord injury.

Significance of effects of spinal cord injury on society is in that spinal cord injuries primarily occur in people in their forties or younger (about 80% of total cases), who actively participate in social activity. Further, the spinal cord injury is largely acquired and thus people with such spinal cord injury are afflicted with heavy physical and mental pains resulting from sudden changes in their circumstances and various limitations in social life. At present, from a medical point of view, currently available treatments are not directed toward active therapy for patients with paraplegia due to spinal cord injury and disorders, but are mainly focused on care to prevent other complications caused by spinal cord injury and disorders (for example, respiratory care, gastrointestinal treatment, prevention of decubitus and thrombosis, and the like).

Conventionally, when functions of organs are partially paralyzed or completely lost, the last option is to perform organ transplantation. In this case, there are problems associated with difficulty in finding the desired organ histologically compatible with the patient, and even following a successful transplantation daily use of immunosuppressant drugs throughout a person's whole life. Immunosuppressant drugs deteriorate the quality of a patient's life and lead to a weakening of immunity, thus resulting in various complications. Further, unfortunately, there is no particular method of transplanting specific tissues or organs available for spinal paraplegic patients, unlike patients with any other intractable diseases.

Meanwhile, stem cells refer to immature cells that still retain the ability to differentiate into other cell types, and when organs or tissues in the body are shocked or damaged by external factors, these cells can migrate to injured sites and differentiate into the corresponding organs or tissues. The stem cell can be broadly divided into an embryonic stem cell, an adult stem cell and a neonatal stem cell, depending on a source thereof. Although it is easy to isolate and cultivate stem cells from bone marrow, there is difficulty in acquisition of the bone marrow and further, at present it is known to be difficult to solve problems associated with an immune rejection occurring when transplanting stem cells to others. Meanwhile, neonatal (umbilical cord) blood is relatively easy to obtain compared with bone marrow, and also, where great numbers of umbilical cord blood units are secured, it is possible to employ umbilical cord blood stem cells that are identical with or most similar to histocompatibility antigens of patients and thereby it is possible to solve problems associated with immune rejection.

Disclosure of Invention Technical Problem

Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to establish a novel cell therapy for a spinal paraplegic patient which is incurable by modern medical techniques, using an umbilical cord blood-derived stem cell having histocompatibility antigens which are identical with those of patients, and to provide a novel therapy involving confirming rapid differentiation of the umbilical cord blood-derived stem cell, which was provided to the patient, into a nerve cell in vitro and thereby inducing neuronal regeneration in the spinal paraplegic patient who cannot fundamentally regenerate neurons.

Technical Solution

In accordance with an aspect of the present invention, the above and other objects can be accomplished by the provision of a composition for transplanting into a spinal cord of a patient for treatment of a spinal paraplegia, comprising mesenchymal stem cells derived from umbilical cord blood, obtained by: adding an anti-coagulant to the umbilical cord blood having a volume of more than 45 ml per unit, which is pure umbilical cord blood obtained within 24 hours after parturition; diluting the resulting mixture of the anti-coagulant and umbilical cord blood with an alpha-minimum essential medium (αMEM), followed by centrifugation to harvest monocytes; and subjecting the obtained monocytes into suspension culture in the αMEM medium containing Stem Cell Factor, GM-CSF (granulocyte-macrophage colony- stimulating factor), G-CSF (granulocyte colony-stimulating factor), IL-3 (interleukin-3) and IL-6 (interleukin-6).

In accordance with another aspect of the present invention, there is provided a composition for transplanting into a spinal cord of a patient for treatment of a spinal paraplegia, comprising mesenchymal stem cells derived from umbilical cord blood, obtained by: thawing cryopreserved umbilical cord blood and adding αMEM (alpha- minimum essential medium) thereto, followed by centrifugation to harvest monocytes; isolating CD133 positive cells from the obtained monocytes; and subjecting the isolated cells into suspension culture in the αMEM containing Stem Cell Factor, GM-CSF (granulocyte-macrophage colony- stimulating factor), G-CSF (granulocyte colony-stimulating factor), IL-3 (interleukin-3) and IL-6 (interleukin-6). In accordance with a further aspect of the present invention, there is provided a method for treatment of spinal paraplegia, comprising: selecting umbilical cord blood in which 6 HLA (Human Leukocyte Antigen) are identical with those of a patient, or one or two HLA are not identical with those of the patient; isolating and culturing stem cells from the selected umbilical cord blood; and transplanting the cultured stem cells into a spinal cord. The present invention has succeed in establishment of a novel cell therapy comprising transplanting stem cells into a spinal cord of a patient under complete exposure of the spinal cord, using an umbilical cord blood-derived stem cell having histocompatibility antigens that are identical with those of a spinal paraplegic patient which is incurable by modern medicine, and induced and confirmed in vitro rapid differentiation of the umbilical cord blood-derived stem cell into a neuronal cell, using a novel combination of culture media. In order to achieve maximized effects, the present invention employs a novel method involving direct transplantation of stem cells into the damaged site using a syringe, instead of using injection of the stem cells into a blood vessel

(vein) which is usually employed in conventional treatments using the stem cells.

Upon considering facts that there is no available active therapy for treating paraplegic patients due to spinal cord disorder or injury, and it is also impossible to perform a transplantation, it can be said that the cell therapy for treating spinal paraplegic patients in accordance with the present invention using the umbilical cord blood-derived stem cell is a monumental landmark in that there is no precedent for it throughout the world. There is no case in which spinal paraplegic patients were treated using any kind of stem cells, throughout the world hitherto, excluding few attempts on animals other than the human.

The cell therapy for treating complete spinal paraplegia using an umbilical cord blood-derived stem cell in accordance with the present invention is based on the condition that the histocompatibility antigens of a stem cell are identical with those of a patient, and thus it is possible to solve the problems of immune rejection by previously preparing the umbilical cord blood-derived stem cell having the histocompatibility antigens that are identical with those of a patient and are thus compatible with the patient. Therefore, the present invention is also particularly significant in that it is possible to treat the patient and it is also possible to improve the quality of patient's life, without common use of immunosuppressant drugs in cell injection for neuronal regeneration of the patient.

The present invention has also confirmed rapid differentiation of the umbilical cord blood-derived stem cell into a neuronal cell by treating the stem cell with a novel combination of reagents in vitro. That is, in conventional differentiation of bone marrow-derived stem cells into neuronal cells, such stem cell differentiation is cAMP (cyclic adenosine monophosphate)-dependent. Whereas, in the case of the umbilical cord blood-derived stem cell in accordance with the present invention, it was possible to achieve differentiation of the umbilical cord blood-derived stem cells into neuronal cells in 24 hours using antioxidants such as DMSO (dimethyl sulfoxide) and BHA (butylated hydroxyanisole), instead of using cell differentiation inducers such as cAMP, 8- bromo-cAMP and forskolin. In this manner, it was confirmed that the umbilical cord blood-derived stem cells in accordance with the present invention differentiate into glial cells and other neuronal cells, appearing at an early stage of neuronal development.

Further, unlike any other embryonic stem cells, the umbilical cord blood- derived stem cell raises absolutely no possibility of teratoma or teratocarcinoma occurrence in the body, and thus it is expected that the cell therapy in accordance with the present invention will give promising hope of possible treatment for spinal paraplegic patients.

Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

Fig. 1 is photographs showing cell characteristics of umbilical cord blood-derived stem cells, thus representing that they are CD13-positive, CD29- positive, SH2-positive and SAMA-positive, respectively; and

Fig. 2 is photographs showing the results of expression of neuronal genes after culturing umbilical cord blood-derived stem cells in a neuronal culture media.

Best Mode

Hereinafter, a cell therapy for treatment of spinal paraplegia using an umbilical cord blood-derived stem cell in accordance with the present invention will be described in more detail with reference to the following Examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and sprit of the present invention.

EXAMPLES

Example 1 : Selection of umbilical cord blood

After determining as to whether 6 Human Leukocyte Antigen (HLA) are identical with those of patients, cryopreserved umbilical cord blood was selected in which 6 HLA were identical with those of patients or one or two HLA were not identical with those of patients.

The human leukocyte antigen is an important factor determining acceptance or rejection of the engrafting of the injected cell, when foreign cells other than autologous cells are injected into the body. In order to determine histocompatibility between a donor and a recipient, HLA is subjected to examination of the total 6 antigens, each examination of which is based entirely on DNA analysis. Such DNA analysis of 6 antigens is to determine on whether A and B regions of HLA class I and a DR region of HLA class II are identical with those of a patient to be implanted.

Example 2-1: Isolation and culture of stem cells from umbilical cord blood

In order to isolate monocytes from umbilical cord blood, the umbilical cord blood was diluted with two-fold volume of αMEM (alpha-minimum essential medium, Jeil Biotech Services, Korea), transferred to a 50 ml Falcon tube and centrifuged at 300xg, at room temperature for 10 min. The separated buffy coat layer was collected, diluted again with two-fold volume of αMEM, overlapped on Ficoll-Hypaque and centrifuged at 300xg, at room temperature for 30 min.

In isolating monocytes from blood, Ficoll-Hypaque, which is a polymer of Ficoll (a polymer of sucrose) and Hypaque (sodium ditrizoate), is largely used.

Ficoll-Hypaque has a specific gravity of 1.077 g/m£, which is heavier than that of monocytes, but lighter than that of red blood cells, thus making it possible to separate from each other by specific gravity difference therebetween. That is, when blood is placed on Ficoll-Hypaque and centrifuged, monocytes gather on the Ficoll-Hypaque.

Monocytes obtained by such a density gradient centrifugation method were placed again in a washing αMEM in which additives were not contained and centrifuged at 200xg, for 10 min, and thereafter, αMEM was discarded except for cells sedimented at the bottom of the Falcon tube, followed by washing. αMEM was added thereto once again, centrifuged at 200xg for 10 min, and thereafter, αMEM was discarded except for cells sedimented at the bottom of the Falcon, followed by washing once again.

Next, to αMEM medium containing an antibiotic (1000 U/M penicillin G, 1000 βglvml Streptomycin sulfate, Gibco-BRL), an anti-fungal agent (0.25 βglvai Amphotericin B), and 2 mM of glutamine (Sigma) were added 20% fetal bovine serum (FBS, Jeil Biotech Services, Korea) and as cell growth factors, Stem Cell Factor (50 ng/m-β), GM-CSF (granulocyte-macrophage colony-stimulating factor; 10 ng/m-6), G-CSF (granulocyte colony-stimulating factor; 10 ngjml), IL-3 (interleukin-3; 10 ng/m#) and IL-6 (interleukin-6; 10 ng/m-ft), and cells were suspended in concentration of 1 x 10⁶/ cm².

After 5 -day culturing, suspended cells were removed from the cultured cell group. When adherent cells were obtained, they were cultured for 25 days in αMEM containing 20% fetal bovine serum and an antibiotic as a culture medium, with complete replacement of culture medium at intervals of 2 days without a washing process . Example 2-2: Isolation and culture of stem cells from crvopreserved umbilical cord blood

Umbilical cord blood units cryopreserved at -196 ^°C were placed and immediately thawed in a water bath at 37^°C . In order to isolate monocytes from the umbilical cord blood, the umbilical cord blood was diluted with two-fold volume of αMEM (alpha-minimum essential medium, Jeil Biotech Services, Korea) and was centrifuged at 300xg for 10 minutes at room temperature. The separated buffy coat layer was collected, diluted again with two-fold volume of αMEM, overlapped on Ficoll-Hypaque and centrifuged at 300xg for 30 minutes at room temperature.

In isolating monocytes from blood, Ficoll-Hypaque, which is a polymer of Ficoll (sucrose polymer) and Hypaque (sodium ditrizoate), is largely used. Ficoll-Hypaque has a specific gravity of 1.077 g/m-C, which is heavier than that of monocytes, but lighter than that of red blood cells, which makes it possible to separate the cells from each other by specific gravity difference therebetween. That is, when blood is placed on Ficoll-Hypaque and centrifuged, monocytes gather on the Ficoll-Hypaque. Monocytes obtained by such a density gradient centrifugation method were additionally washed twice with a washing αMEM in which additives were not included.

From the thus-obtained monocytes, CD 133 positive cells were selected using an Isolation kit (Miltenyi Bioteck, Germany) as follows: 100 μi of a blocking reagent was added to monocytes so as to remove non-specific bonding, and then homogeneously mixed with 100 μi of a CD133/Microbead to a total volume of 500 μi. The resulting mixture was then cultured at 4^°C for 30 minutes. The culture was added with a ten-fold volume of PBS (D-phosphate buffered saline, Jeil Biotech Services, Korea), centrifuged at 300xg for 10 minutes, and thereafter, PBS was discarded to obtain the cells adhered to the tube.

The cells were resuspended in 500 μi of PBS. After the column of Isolation kit was previously washed with 3 ml of a PBS buffer, the resuspended cells were loaded and maintained in the column for more than 15 minutes. The column, after rinsed with PBS four times, was removed from the kit and then added with an appropriate amount of PBS in a tube, followed by flushing using a plunger, thereby selecting positive cells.

Next, 20% fetal bovine serum (FBS, Jeil Biotech Services, Korea), and as cell growth factors, Stem Cell Factor (50 ngM), GM-CSF (granulocyte- macrophage colony-stimulating factor; 10 ng/m£), G-CSF (granulocyte colony- stimulating factor; 10 ng/mi), IL-3 (interleukin-3; 10 ng/m#) and IL-6 (interleukin- 6; 10 ng/m£) were added to αMEM containing antibiotics (1000 U/m# of penicillin G, 1000 βg/ml of streptomycin sulfate, Gibco-BRL), an anti-fungal agent (0.25 βg/ml amphotericin B) and 2 mM glutamine (Sigma) and the selected cells were suspended therein to a concentration of 1 x 10⁶ cells/cm'. After five-day culturing, suspended cells were removed from the cultured cell group. When adherent cells were obtained, they were cultured for 25 days in αMEM containing 20% fetal bovine serum and antibiotics as a culture medium, with complete replacement of a culture medium at intervals of 2 days without a washing process.

Example 3: Differentiation of umbilical cord blood-derived stem cells into nerve cells in vitro

0.05% trypsin-EDTA was added to stem cells, which were cultured for two weeks in Example 2, so that adherent cells were suspended. Then, suspended cells were further cultured in 20% low-glucose DMEM to a cell density of 1 x 10⁶ cells/cm². Cells were cultured for two days and a culture medium was discarded. Adherent cells were washed with low-glucose DMEM. After washing cells, production of neuronal cells was induced in low-glucose DMEM containing 20% fetal bovine serum and 10 ng of bFGF as a culture medium. Thereafter, adherent cells were washed once with D-PBS (D- phosphate buffered saline) which was previously warmed to 37 ^°C, and neuronal differentiation was induced in low-glucose DMEM containing 25 mM KCl, 2% dimethyl sulfoxide (DMSO) and 100 μ M butylated hydroxyanisole (BHA), 5 βg/ml insulin, 100 μg/ml transferrin, 2O nM progesterone, 100 μM putrescine, 20 nM sodium selenite and 20 ng/mt bFGF.

Fig. 1 is photographs showing cell characteristics of umbilical cord blood-derived stem cells, thus representing that cells are CD13-positive, CD29- positive, SH2 -positive and SAMA-positive, respectively. Fig. 2 is photographs showing the results of expression of neuronal genes after culturing umbilical cord blood-derived stem cells in a neuronal culture media. As can be seen, expressed cells exhibited neuronal morphology in 24 hours. Further, it was possible to confirm expression of Tujl, TrkA, GFAP and CNPase, which are marker proteins of neuronal differentiation.

Clinical trial example: Treatment results of spinal paraplegic patient

1. Medical history of the spinal paraplegic patient (1) Patient: Woman, age of 37 (2) Pathogenic cause: Spinal paraplegia caused by tumbling down in a creek 20 years ago

(3) Name of a disease: Complete spinal paraplegia, complete paralysis downward from the 9^l thoracic vertebra (left) and the 12^th thoracic vertebra

(right)

2. Surgical therapy

In a manner that stem cells were directly transplanted into the 9^th thoracic vertebra under complete exposure of a spinal cord, using umbilical cord blood- derived stem cells having histocompatibility antigens that are identical with those of patients, surgical operation was conducted for 5 hours from 8 a.m. to 1 p.m., on

October 12, 2004.

More specifically, stem cells, cultured for about 3 weeks, were placed in 0.05% trypsin-EDTA and reacted at room temperature for 5 min. Then, D-PBS was added thereto and the resulting mixture was centrifuged at 300xg for 10 min.

After centrifugation, 5 ml of physiological saline was added to the remaining cells in a test tube and were centrifuged at 300xg for 10 min. This step was repeated thrice such that stem cells were washed clean to the maximum extent possible, thus being free from other composition ingredients. Finally, stem cells remaining in the test tube were adjusted to a cell density of a total of 5 x 10⁷ cells in 6 ml of physiological saline, and the thus re-suspended cells were directly injected to the affected site using a syringe.

After transplanting stem cells, the exposed spinal cord was sutured again by surgical therapy, and examination was made on neurological changes in the lower half of patient's body. 3. Surgical results

Evaluation of the Dermatomal Somatosensory Evoked Potentials (DSEP): Dermatomal Somatosensory Evoked Potential (DSEP) test is a method of recording electrical response by applying electrical stimuli to sensory neurons at ends of the upper or lower extremities and inserting a recording electrode into the cerebral cortex in which the stimulated neurons are received. This test method can be used to diagnose which parts ranging from hands and feet to the cerebral sensory cortex there is abnormality or amelioration by stimulating somatosense of hands and feet and then analyzing electrical signals generated in the cerebral sensory cortex.

The results of Dermatomal SEP before and after transplantation are shown in Tables 1 and 2 below. Table 1 represents the results of the right side, and Table 2 represents the results of the left side. For reference, the spinal column (also called the backbone) consists of a series of 31 individual segments and bones, i.e. the cervical vertebrae (C1-C9), the thoracic vertebrae (Tl -T 12), the lumbar vertebrae (L1-L5) and the coccyx or tailbones (S1-S5), from the top to the bottom.

Table 1

Table 2

As can be seen from Tables 1 and 2 above, it can be confirmed through the results of dermatomal SEP for the right side with respect to the passage of time after transplantation that the first lumbar vertebra exhibited no response on Oct. 11 , but surprisingly exhibited sudden appearance of sensation and finally neuronal regeneration on Nov. 1. In addition, it can also be confirmed that the second lumbar vertebra (L2) initially exhibited no response, but showed neuronal regeneration on Nov. 1. In the case of the left side neurons, it can be confirmed that 12^th thoracic vertebra (T 12) initially exhibited no response, but began to show some response exactly 3 days later, i.e. on Oct. 19, and the first (Ll) and second (L2) lumbar vertebrae also exhibited neuronal regeneration on Nov. 1.

Industrial Applicability

As described above, a cell therapy for treatment of complete spinal paraplegia utilizing an umbilical cord blood-derived stem cell in accordance with the present invention is based on the condition that the histocompatibility antigens of a stem cell are identical with those of a patient to be transplanted and thus can solve the problems of immune rejection without use of immunosuppressant drugs. Further, it is advantageous in that unlike the cases in which any other embryonic stem cells are employed, the cell therapy in accordance with the present invention using the umbilical cord blood stem cell presents no possibility of teratoma or teratocarcinoma occurrence in the body, and thus it is expected that the cell therapy in accordance with the present invention will give promising hope of a possible treatment for spinal paraplegic patients.

Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

1. A composition for transplanting into a spinal cord of a patient for treatment of a spinal paraplegia, comprising mesenchymal stem cells derived from umbilical cord blood, obtained by: adding an anti-coagulant to the umbilical cord blood having a volume of more than 45 ml per unit, which is pure umbilical cord blood obtained within 24 hours after parturition; diluting the resulting mixture of the anti-coagulant and umbilical cord blood with an alpha-minimum essential medium (αMEM), followed by centrifugation to harvest monocytes; and subjecting the obtained monocytes into suspension culture in the αMEM medium containing Stem Cell Factor, GM-CSF (granulocyte-macrophage colony- stimulating factor), G-CSF (granulocyte colony-stimulating factor), IL-3 (interleukin-3) and IL-6 (interleukin-6).

2. A composition for transplanting into a spinal cord of a patient for treatment of a spinal paraplegia, comprising mesenchymal stem cells derived from umbilical cord blood, obtained by: thawing cryopreserved umbilical cord blood and adding αMEM (alpha- minimum essential medium) thereto, followed by centrifugation to harvest monocytes; isolating CD133 positive cells from the obtained monocytes; and subjecting the separated cells into suspension culture in the αMEM containing Stem Cell Factor, GM-CSF (granulocyte-macrophage colony- stimulating factor), G-CSF (granulocyte colony-stimulating factor), IL-3 (interleukin-3) and IL-6 (interleukin-6).

3. A method for treatment of spinal paraplegia, comprising: selecting umbilical cord blood in which 6 HLA (Human Leukocyte

Antigen) are identical with those of a patient, or one or two HLA are not identical with those of the patient; isolating and culturing stem cells from the selected umbilical cord blood; and transplanting the cultured stem cells into spinal cord.