ES2550456T3 - Use of a composition containing mesenchymal stem cells derived from human umbilical cord blood to induce differentiation and proliferation of neural precursor cells or neural stem cells to neural cells - Google Patents

Abstract

Use of a composition comprising mesenchymal stem cells derived from umbilical cord blood (UCB-MSC) to induce differentiation and proliferation of neural precursor cells or neural stem cells to neural cells in vitro, comprising cocultivating UCB-MSC with neural precursor cells or neural stem cells

Description

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DESCRIPTION

Use of a composition containing mesenchymal stem cells derived from human umbilical cord blood to induce differentiation and proliferation of neural precursor cells or neural stem cells to neural cells

Field of the Invention

The present invention relates to uses of a composition comprising mesenchymal stem cells derived from human umbilical cord blood to induce differentiation and proliferation of neural precursor cells or neural stem cells to neural cells.

Background of the invention

Stroke, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic disease of the central nervous system and spinal cord injury disease involve dysneuria caused by nerve cell injury, and have been treated usually with medication or surgical operation, which can seriously damage normal cells.

Recently, it has been recognized that a cell replacement therapy in which normal cells are transplanted to replace damaged or destroyed cells is effective for such diseases, and stem cells, in particular, that can be differentiated and proliferated to give desired tissues are being intensively studied.

Stem cells are non-specialized cells that can proliferate unlimitedly in the undifferentiated phase and can differentiate into various tissues in response to specific stimuli.

Neural stem cells, from which neurons and / or glial cells such as astrocytes, oligodendrocytes and / or Schwann cells are formed, are also undifferentiated cells that have potential for self-reproduction. They differ in neural cells, for example neurons or glial cells through neural precursor cells or glial precursor cells.

It is known that mesenchymal stem cells, which differ in bone, cartilage, adipose tissue, muscle, tendon, ligament, neural tissue and others, are viable for cell replacement therapy. Mesenchymal stem cells have been obtained primarily from the bone marrow, but such mesenchymal stem cells provide only limited applications due to their restrictive potential for differentiation and proliferation. In addition, complicated and often painful operations that consist of several stages for such cell replacement therapy should be performed, in addition to the problem of finding a donor that has histocompatibility antigens identical to those of a patient to exclude the graft-versus-host reaction during bone marrow transplant.

In recent years, umbilical cord blood has become a target for researchers due to its high concentrations of stem cells. Several trials have been conducted to treat blood diseases by transplanting umbilical cord blood to a patient, and umbilical cord blood banks have been established, which conserve umbilical cord blood in frozen form until use, for autologous transplant therapy.

Unlike the bone marrow, umbilical cord blood can be obtained by a simple operation from an umbilical cord and causes little graft reaction against host. For these reasons, worldwide studies have recently been conducted for the clinical application of umbilical cord blood.

The present inventors have also extensively studied mesenchymal stem cells derived from umbilical cord blood and have found that they can induce differentiation and proliferation of neural precursor cells or neural stem cells to neural cells.

Summary of the invention

Therefore, it is an object of the present invention to provide the use of a composition comprising mesenchymal stem cells derived from umbilical cord blood (UCB-MSC) to induce differentiation and proliferation of neural precursor cells or neural stem cells to neural cells in vitro , which comprises culturing UCB-MSC with neural precursor cells or neural stem cells.

According to another aspect of the present invention, there is provided a method for inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells in vitro, comprising cocultivating mesenchymal stem cells derived from umbilical cord blood with neural precursor cells or cells. neural stem cells

Also disclosed herein is a composition to induce differentiation and proliferation of neural precursor cells or neural stem cells to neural cells, which comprises mesenchymal stem cells derived from umbilical cord blood as an active component.

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According to a still further aspect of the present invention, UCB-MSC are provided for use in the treatment of a nerve injury disease by inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells.

Brief description of the drawings

The foregoing and other objects and features of the present invention will be apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which show respectively:

Figure 1: a diagram of a Transwell chamber used to cocultivate mesenchymal stem cells derived from umbilical cord blood and neural precursor cells;

Figure 2: Photographs showing the differentiation and proliferation of NG108-15 (NG108) observed with a phase contrast microscope (x100), 4 and 7 days after culture of NG108-15 alone or co-culture with the same stem cells mesenchymal cells derived from umbilical cord blood, respectively;

Figure 3: Photographs showing the result of immunostaining for tubulin beta-Ill, an early marker for neuronal differentiation, 7 days after the culture of NG108-15 alone or co-culture with them of mesenchymal stem cells derived from umbilical cord blood, respectively;

Figure 4: Photographs showing the differentiation and proliferation of NG108-15 observed with a phase contrast microscope (x100), 7 days after the culture of NG108-15 cells alone, supplemented with cAMP or in coculture with stem cells derived from mesenchymal stem cells. umbilical cord blood obtained from two different individuals (hUCB-MSC-1 and hUCB-MSC-2), respectively;

Figure 5: Photographs showing the differentiation and proliferation of neural stem cells derived from the cerebral cortex of a fetal mouse observed with a phase contrast microscope (x 100), 7 days after co-culture of neural stem cells with hUCB-MSC -1 and hUCB-MSC-2 of various concentrations, respectively;

Figure 6: Photographs showing the result of immunostaining for tubulin beta-Ill and microtubule-associated protein 2 (MAP2), early markers of neuronal differentiation, 7 days after the culture of neural stem cells derived from the cerebral cortex of the fetal mouse alone or co-culture with them of mesenchymal stem cells derived from umbilical cord blood, respectively;

Figure 7: a graph showing the number of viable cells evaluated by trypan blue staining, 7 days after co-culture of NG108-15 with mesenchymal stem cells derived from umbilical cord blood of various concentrations; Y

Figure 8: a graph showing the number of viable cells evaluated by trypan blue staining, 7 days after co-culture of neural stem cells, derived from the cerebral cortex of the fetal mouse, with mesenchymal stem cells derived from umbilical cord blood from various concentrations.

Detailed description of the invention

The composition for inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells typically comprises mesenchymal stem cells derived from umbilical cord blood as an active component.

As used herein, the term "umbilical cord blood" refers to blood drawn from the umbilical cord vein that joins the placenta of a mammal with a newborn from it.

The term "mesenchymal stem cells derived from umbilical cord blood" as used herein refers to mesenchymal stem cells that are isolated from umbilical cord blood of a mammal, preferably a human being.

The term "a disease due to nerve injury" as used herein refers to a disease that is accompanied, among others, by behavioral dysfunction due to damaged sensory or motor nerves. Exemplary nerve injury diseases include stroke, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, amyotrophic lateral sclerosis, traumatic central nervous system disease, and spinal cord injury disease.

The term "treat" refers to: preventing the manifestation of a disease or disorder not yet diagnosed in an animal, preferably a mammal, most preferably a human being, who is prone to acquire such disease or disorder; or inhibit the development of a disease due to nerve injury.

The term "a neural cell" as used herein refers to a neuron of the central or peripheral nervous system, and / or a glial cell such as an astrocyte, an oligodendrocyte and / or a Schwann cell.

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In the isolation of a monocyte comprising mesenchymal stem cells from umbilical cord blood, a common method such as the Ficoll-Hypaque density gradient method can be employed. Specifically, said method comprises the steps of collecting umbilical cord blood from the umbilical vein after delivery until the placental abruption; centrifuge umbilical cord blood with a Ficoll-Hypaque gradient to obtain monocytes; and remove contaminants from them. The monocytes obtained can be subjected to isolation of mesenchymal stem cells thereof, or to deep-freezing for long-term safe maintenance until use.

Isolation of mesenchymal stem cells from monocytes derived from umbilical cord blood can be performed by the method of Yang SE et al. (Yang SE et al., Cytotherapy, 6 (5): 476-486, 2004). Specifically, monocytes are suspended in a medium containing 5 to 30% by weight, preferably 5 to 15% by weight of fetal bovine serum (FBS), including conventional medium one such as DMEM, α-DMEM, basal medium of Eagle or RPMI 1640. Then, the cells in the suspension are divided into media having the same composition described above and grown in an incubator with 5% CO2 at 37 ° C. When cultured cells form a monolayer, mesenchymal stem cells are observed that have a spindle shape. Then, the mesenchymal stem cells are repeatedly subcultured until the cells are sufficiently amplified.

According to the present invention, both differentiation and proliferation of neural precursor cells or neural stem cells to neural cells can be induced by co-cultivation of mesenchymal stem cells derived from umbilical cord blood with neural precursor cells or neural stem cells. Specifically, mesenchymal stem cells derived from umbilical cord blood are simultaneously effective not only to induce differentiation of neural precursor cells or neural stem cells to give neural cells, but also to sustain and reinforce such effects through an increase in the number of the neural cells, to enhance their therapeutic effects.

Therefore, the present invention provides uses of mesenchymal stem cells derived from umbilical cord blood or a composition comprising the cells to induce differentiation of neural precursor cells or neural stem cells to neural cells and the proliferation of the resulting neural cells.

Mesenchymal stem cells derived from umbilical cord blood or a composition comprising the cells for cytotherapy of a patient suffering from nerve injury diseases, for example, stroke, Parkinson's disease, Alzheimer's disease, Pick's diseases, Pick's disease, can be used Huntington, amyotrophic lateral sclerosis, traumatic diseases of the central nervous system and spinal cord injury disease, preferably stroke and spinal cord injury disease.

The composition disclosed herein may further comprise a pharmaceutically acceptable additive.

A pharmaceutical formulation can be prepared in a unit dosage form using the composition disclosed herein according to conventional procedures in the art. A formulation for parenteral administration such as an injection or a topical dosage form is preferable. The pharmaceutical formulation may further include pharmaceutically acceptable additives, for example, fillers, expanders, binding agents, wetting agents, disintegrants, diluents such as surfactants and other excipients.

The pharmaceutical formulation can be administered parenterally according to conventional procedures in the art, for example, by direct injection into a region of injury as well as injection into the cerebrospinal fluid, for example, lumbar puncture and injection into the parenchyma, vein or artery. Preferably, it can be administered by direct injection into a peripheral or opposite region of the spinal cord or brain injury region. In addition, the clinical method of Douglas Kondziolka (Douglas Kondziolka, Pittsburgh, 1998) can be used to administer the inventive pharmaceutical formulations in a region of injury. Specifically, an incision is made in the skull of a subject to make a hole that has a diameter of 1 cm and a suspension of mesenchymal stem cells is injected into HBSS (Hank's balanced saline solution) in the hole using a long needle syringe and a stereotactic framework.

A typical dose of mesenchymal stem cells can range between 1x105 and 1x107 cells / kg body weight / injection, preferably between 5x105 and 5x106 cells / kg body weight / injection, which can be administered in a single dose or in divided doses. In addition, it should be understood that the amount of the active component actually administered to a given patient must be determined in view of various relevant factors including the amount of neural cells that are to be differentiated and proliferated, the route of administration chosen and the body weight, age and The sex of an individual patient.

The present invention also provides a method for inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells in vitro, comprising cocultivating mesenchymal stem cells derived from umbilical cord blood with neural precursor cells or stem cells

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Neurals Co-culture can be carried out by mixing mesenchymal stem cells derived from umbilical cord blood with neural precursor cells or neural stem cells at a ratio of 1: 0.1 to 1:10, preferably 1: 1 to 1: 2 based on their cell number and cutting the cell mixture into a conventional cell culture medium such as DMEM, α-DMEM, α-MEM, Eagle's basal medium and RPMI 1640. The culture medium may further comprise an antibiotic, for example, gentamicin, and / or 5 to 15% by weight of FBS. The cultivation period can range from 5 to 10 days.

The present invention also provides the use of mesenchymal stem cells derived from umbilical cord blood for the preparation of a pharmaceutical composition for treating a disease by nerve injury by inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells. The administration of mesenchymal stem cells derived from umbilical cord blood or a composition comprising them to the nerve cell injury region of a subject in need of treatment of nerve injury disease is disclosed herein. The subject can be a mammal including a human being.

When administered in a therapeutically effective amount, the mesenchymal stem cell derived from umbilical cord blood induces not only differentiation of neural precursor cells or neural stem cells from the central or peripheral nervous system to neural cells, but also the proliferation of the resulting neural cells , thereby resulting in the recovery of neural functions and the treatment of such disease due to nerve damage. The treatment effect of the mesenchymal stem cell derived from umbilical cord blood is greatly enhanced and sustained for a long period of time by its ability to proliferate regenerated neural cells.

The following examples and test examples are provided for the purpose of illustration only, and are not intended to limit the scope of the invention.

Example 1: Isolation and culture of mesenchymal stem cells derived from umbilical cord blood

(Stage 1) Acquisition of umbilical cord blood (UCB)

A sample of UCB was obtained from the umbilical vein of a parturient woman with her consent. Specifically, a 16 gauge needle from a UCB collection bag containing 44 ml of CPDA-1 anticoagulant (GREEN CROSS) was inserted into the umbilical vein to allow the flow of UCB into the bag. The collected blood was processed within 48 hours and the cell survival rate was above 90%.

(Stage 2) Isolation and amplification of mesenchymal stem cells

The UCB obtained in step 1 was subjected to centrifugation using a gradient of Ficoll-Hypaque (density: 1,077 g / ml, Sigma) to obtain monocytes. The monocytes were then washed several times to remove impurities and suspended in a minimum basal medium containing 5 to 15% by weight of FBS (HyClone) (α-MEM, Gibco BRL). Subsequently, a previously measured amount of the suspension was added to the same medium as above and grown in a 5% CO2 incubator at 37 ° C, while the medium was exchanged for a new batch of medium twice a week. When the cells in culture formed a monolayer, the generation of mesenchymal stem cells that had a spindle shape with a microscope was confirmed. The mesenchymal stem cells thus formed were repeatedly subcultured until the cells were sufficiently amplified (Yang SE et al., Cytotherapy, 6 (5): 476-486, 2004).

Example 2: Culture of NG108-15 (NG108)

A mouse brain-derived cell, NG108-15 (neuroblastoma X glioma hybrid) (ATCC, cat. No. ATCCCRL-HB-12317), which had physiological and morphological characteristics similar to a neural precursor cell was cultured in DMEM (Dulbecco's modified Eagle's medium) (4 mM / l glutamine, 4.5 g / l glucose, 4.0 mg / l pyridoxine-HCl, 0.1 mM hypoxanthine-guanine, 400 nM aminopterin, 0.016 mM thymidine, FBS 5 to 15% by weight).

Example 3: Neural Stem Cell Culture

Neural stem cells derived from the cerebral cortex of a fetal mouse (Chemicon, cat. No. SCR029) were cultured in a basal medium of neural stem cells (FGF-2 20 ng / ml, EGF 20 ng / ml and heparin 2 mg / ml)

Example 4: Co-culture of mesenchymal stem cells derived from umbilical cord blood and NG108-15 (I)

Mesenchymal stem cells derived from human umbilical cord blood (hUCB-MSC) from Example 1 were co-cultured with NG108-15 from Example 2 (hUCB-MSC: NG108-15 = 1: 1) using a Transwell chamber (Figure 1) and the culture medium of example 2. As a control group, NG108-15 were grown alone in the culture medium of example 2. As shown in Figure 1, the Transwell chamber was composed of lower and upper compartments that were separated from one another by a microporous membrane that had pores of 1 µm. The hUCB-MSC were placed in the upper compartment, and the NG108-15 in the lower compartment.

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The differentiation of NG108-15 was observed with a phase contrast microscope (X 100) at 4 and 7 days. As shown in Figure 2, NG108-15 co-cultivated with hUCB-MSC differentiated into a typical mature neuron-like cell form in which the cells extend into long branches and differentiate to have a spindle shape.

It was further confirmed that the differentiated cells were neuron-like cells at 7 days using immunostaining for tubulin-beta III, an early marker of neuronal development. Specifically, hUCB-MSC, NG108-15 and a mixture thereof were respectively grown on a coverslip, and blocked by adding them to 10% normal goat serum containing 0.3% Triton X-100 for 1 hour at temperature. ambient. The 1st antibody used in immunostaining, a monoclonal mouse antibody anti-tubulin-beta III conjugated to phycoerythrin (Chemicon), was diluted one hundred times with goat serum and added thereto. The mixture was kept overnight at 4 ° C, and the resulting mixture was washed three times (each time for 5 minutes) with 0.01 M PBS.

As shown in Figure 3, NG108-15 co-cultured with the mesenchymal stem cells of Example 1 presented a different response to immunostaining for tubulin-beta III, verifying that the differentiated cells were neuron-like cells.

Example 5: Co-culture of mesenchymal stem cells derived from umbilical cord blood and NG108-15 (II)

Mesenchymal stem cells derived from human umbilical cord blood were obtained by the method of Example 1 from two different individuals (hUCB-MSC-I and hUCB-MSC-2). NG108-15 of Example 2 was cultured alone or co-cultured with hUCB-MSC-1 or hUCB-MSC-2 according to the method of Example 4 for 7 days. Differentiation and proliferation of the cells were observed with a phase contrast microscope (x 100).

As a comparative group, 1 mM cAMP was added that induces differentiation of NG108-15 to neuron-like cells (NeuroReport 9, 1261-1265, 1998) to the culture medium of NG108-15.

As shown in Figure 4, NG108-15 co-cultured with mesenchymal stem cells differentiated into a form of cells similar to mature neurons. There was no significant difference between hUCB-MSC-1 and hUCB-MSC-2 in their differentiation induction activities.

Example 6: Co-culture of mesenchymal stem cells derived from umbilical cord blood and neural stem cells (I)

The neural stem cells of Example 3 were cultured alone (control group) or co-cultured with hUCB-MSC-1 or hUCB-MSC-2 of Example 5 in the culture medium of Example 3, using the Transwell chamber. The hUCB-MSC were placed in the upper compartment and the neural stem cells in the lower compartment.

HUCB-MSC-1 and hUCB-MSC-2 were respectively added to the medium at the concentration of 500, 1000, 2000, 4000 and 6000 cells / cm2, respectively, and the neural stem cells were cocultured with the concentration of 2000 cells / cm2 . Differentiation and proliferation of the cells were observed with a phase contrast microscope (x100) (Figure 5) at 7 days.

As shown in Figure 5, neural stem cells co-cultured with mesenchymal stem cells differentiated into a form of mature neurons. There was no significant difference between hUCB-MSC-1 and hUCB-MSC-2 in their differentiation induction activities. In addition, the degree of differentiation and proliferation of neural stem cells was directly proportional to the concentration of mesenchymal stem cells with which they were cocultured.

Example 7: Co-culture of mesenchymal stem cells derived from umbilical cord blood and neural stem cells (II)

Neural stem cells from example 3 were cultured alone (control group) or co-cultured with hUCB-MSC from example 1 (hUCB-MSC: neural stem cells = 1: 1) in the culture medium of example 3, using a chamber Transwell The hUCB-MSC were placed in the upper compartment and the neural stem cells in the lower compartment.

Immunostaining was performed at 4 and 7 days using the method of example 4 for tubulin-beta III and microtubule-associated protein 2 (MAP2), early markers of neuronal development, so that the differentiated cells were confirmed to be neurons .

As shown in Figure 6, the neural stem cells co-cultured with the mesenchymal stem cells of Example 1 presented a different response to immunostaining for tubulin-beta III and MAP2, verifying that the differentiated cells were neurons.

Example 8: Co-culture of mesenchymal stem cells derived from umbilical cord blood with neural precursor cells or neural stem cells

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NG108-15 of example 2 alone (control group) was cultured or co-cultured with hUCB-MSC of example 1, and the neural stem cells of example 3 alone (control group) were cultured or cocultured with hUCB-MSC-1 or hUCB-MSC-2 of Example 5, according to the methods of Examples 4 and 6, respectively. The number of viable cells was counted at 7 days using trypan blue staining (Figures 7 and 8).

5 As shown in Figures 7 and 8, the numbers of NG108-15 and neural stem cells increased in proportion to the concentration of the mesenchymal stem cells with which they were cocultured, which implies that mesenchymal stem cells derived from Umbilical cord blood can be simultaneously effective not only to induce differentiation of neural precursor cells or neural stem cells to neural cells, but also to sustain and reinforce such effects through increasing cell numbers

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Claims (4)

1. Use of a composition comprising mesenchymal stem cells derived from umbilical cord blood (UCB-MSC) to induce differentiation and proliferation of neural precursor cells or neural stem cells to neural cells in vitro, comprising cocultivating UCB-MSC with precursor cells 5 neural or neural stem cells.

2.

Method for inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells in vitro, comprising cocultivating mesenchymal stem cells derived from umbilical cord blood with neural precursor cells or neural stem cells.

3.

Method according to claim 2, wherein the ratio of mesenchymal stem cells derived from

10 umbilical cord blood with respect to neural precursor cells or neural stem cells employed is in the range of 1: 0.1 to 1:10. 4. Mesenchymal stem cells derived from umbilical cord blood (UCB-MSC) for use in inducing differentiation and proliferation of neural precursor cells or neural stem cells to neural cells in a patient suffering from a nerve injury disease. 15 5. Mesenchymal stem cells derived from umbilical cord blood for use according to claim 4, wherein the nerve injury disease is stroke, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease, lateral sclerosis amyotrophic, traumatic disease of the central nervous system or disease due to spinal cord injury. twenty 8