WO2008088118A1 - Neuronal regeneration material screening method by ex vivo model - Google Patents

Neuronal regeneration material screening method by ex vivo model Download PDF

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WO2008088118A1
WO2008088118A1 PCT/KR2007/005107 KR2007005107W WO2008088118A1 WO 2008088118 A1 WO2008088118 A1 WO 2008088118A1 KR 2007005107 W KR2007005107 W KR 2007005107W WO 2008088118 A1 WO2008088118 A1 WO 2008088118A1
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spinal cord
neuronal regeneration
slices
screening method
mesenchymal stem
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PCT/KR2007/005107
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French (fr)
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Mi-Sook Chang
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Seoul National University Industry Foundation
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    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
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    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/285—Demyelinating diseases; Multipel sclerosis

Definitions

  • the present invention relates to a neuronal regeneration material screening method using ex vivo models, more precisely a screening method for a material that is able to regenerate neuronal cells by using the spinal cord tissue slices.
  • Mesenchymal stem cells were first found in the mouse bone marrow cells, which were in the form of fibroblasts.
  • mesenchymal stem cells are classified into several mesodermal series and these cells can be differentiated into various cells, that is mesenchymal stem cells are characterized by their diversity in differentiation into osteoblasts, adipocytes, endothelial cells and neuronal cells .
  • ex vivo model is now in the center of our concern as an alternative experimental model overcoming disadvantages of both in vivo and in vitro models.
  • the ex vivo model such as organotypic slice cultures has been used for the study of spinal cord lesion model such as ischemia and cytotoxicity (Krassioukov et al., J Neurotrauma 19:1521-1529, 2002).
  • the spinal cord slice culture has dorsal horn and ventral horn, distinguished from each other, and keeps its excellent form as a whole, making the confirmation of neuron distribution easy.
  • Lysolecithin is a lipid containing detergent-like protein and has a similar activity with a membrane soluble agent exhibiting myelinated cell-specific toxicity to cause demyelination. That is, lysolecithin inserted in myelinated fiber such as the spinal cord in vivo causes demyelination, leading to the spinal cord injury.
  • loss of oligodendrocytes, along with multiple scierosis has been known to be a cause of various adult demyelination associated diseases, confirming the association of oligodendrocytes and myelination of the spinal cord.
  • oligodendrocytes When the precursor cells of human oligodendrocytes were transplanted into the adult mouse brain damaged by lysolecithin, the brain cells were fast developed like oligodendrocytes and the exposed axons were myelinated even though not as firm as oligodendrocytes and with comparatively low efficiency (Goldman, Nat Biotechnol 23:862-871. 2005). These results suggest that axons demyelinated by lysolecithin can be remyelinated by the transplantation of mesenchymal stem cells.
  • the present inventors confirmed that neuronal regeneration by remyelination of damaged axons in spinal cord slices could be induced after transplantation of bone marrow-derived mesenchymal stem cells into those slices which had previously been demyelinated by a toxic compound. Furthermore, the present inventors completed this invention by confirming that directional neuronal regeneration could be effectively applied as a novel treatment method for neurodegenerative diseases.
  • the present invention provides a screening method for a neuronal regeneration material by using spinal cord slices.
  • the present invention also provides a method for neuronal regeneration of the demyelinated spinal cord containing the step of administering bone marrow-derived mesenchymal stem cells into the injured spinal cord of a subject .
  • the present invention further provides a cell composition for neuronal regeneration of the demyelinated spinal cord comprising bone marrow-derived mesenchymal stem cells.
  • the method for neuronal regeneration in the demyelinated area by transplanting bone marrow-derived mesenchymal stem cells and the method for screening a neuronal regeneration material using ex vivo models of spinal cord live tissue slices of the present invention can be effectively applied as a novel treatment method for neurodegenerative diseases.
  • Fig. 1 is a set of photographs showing the demyelinations of spinal cord slices by lysolecithin: a: Transmittance; b: H & E staining; and, c: Immunofluorescence using MBP antibody.
  • Fig. 2 is a set of photographs illustrating neuronal regeneration of the demyelinated spinal cord slices by the transplantation of mesenchymal stem cells: a: Non-demyelinated group (control group); b: Demyelinated group (Lyso+PBS) ; and, c: Remyelinated group (Lyso+MSC) .
  • Fig. 3 is a set of photographs illustrating the immunofluorescence of cell nuclei of neurofilaments (150 kDa) to confirm neuronal regeneration of the demyelinated spinal cord slices by the transplantation of mesenchymal stem cells: a: 1 week later; and, b: 2 weeks later.
  • Fig. 4 is a set of photographs illustrating the immunofluorescence of neurofilaments (150 kDa) to confirm neuronal regeneration of the dernyelinated spinal cord slices by the transplantation of mesenchymal stem cells: a: 1 week later; and, b: 2 weeks later.
  • Fig. 5 is a set of graphs illustrating the increase of the expression of neurofilaments (150 kDa) by neuronal regeneration of the demyelinated spinal cord section by the transplantation of mesenchymal stem cells: a: 1 week later; and, b: 2 weeks later.
  • Fig. 6 is a set of photographs illustrating the immunofluorescence using Pl and CNPase to confirm neuronal regeneration of the demyelinated spinal cord slices by the transplantation of mesenchymal stem cells: a: 1 week later; and, b: 2 weeks later.
  • the present invention provides a screening method for a neuronal regeneration material comprising the following steps: 1) Culturing tissue slices obtained from the spinal cord;
  • step 2) Damaging the tissue slices in course of the culture, of step 1) by treating with a toxic compound; 3) Administering neuronal regeneration material candidates to the damaged tissue slices; and
  • the experiments designed with animal are classified as the in vivo model targeting an individual animal, the in vitro model targeting a cell culture and the ex vivo model targeting a live tissue.
  • the disadvantage of in vivo model is that the regulation of various endogenous factors is difficult and thereby the result can be misinterpreted.
  • the in vitro model has also disadvantages of not-guaranteeing the activity of biomaterials .
  • the live tissue slices obtained from spinal cord of the invention can be cultured for 3 weeks since their separation is carried out promptly by using a tissue chopper. Thus, they can be used as an ex vivo system with advantages from both in vivo and in vitro systems.
  • the system established in the present invention facilitates the study of acute and sub-acute pathophysiology in addition to enabling the regulation of associated environments with the goal of the experiment. This system has excellent reproducibility, compared with other animal models, and is economical.
  • the tissue slices of the present invention can survive approximately three weeks under the proper culture condition. Thus, they can be effectively applied for screening of novel materials involved in the regeneration of damaged spinal cord tissues. More particularly, after damaging neurons of the tissue slices artificially, a candidate molecule expected to induce the regeneration of the damaged neuron can be adminstered and the slices can be cultured for 2 - 3 weeks. Then, the regeneration level and characteristics can be observed to investigate the usability of the novel candidate material.
  • the thickness of the tissue slices is 300 ⁇ m - 500 ⁇ m, more preferably 400 ⁇ m.
  • the toxic compound is any substance capable of inducing demyelination of the spinal cord axon, and preferably lysolecithin.
  • the neuronal regeneration effect is confirmed by measuring the expression of a neuroglia cell marker protein labeled by immunofluorescence, observing neuronal regeneration by histological method, and measuring apoptotic cells by TUNEL assay.
  • the injury includes every neuronal injury in the spinal cord and preferably the injury caused by demyeli nation.
  • the candidate for neuronal regeneration is selected from the group consisting of compounds, growth factors, cytokines, mesenchymal stem cell originated factors, extracellular matrix proteins, proteins involved in the axonal outgrowth and factors involved in intracellular signal transduction.
  • the present invention also provides a method for neuronal regeneration of the demyelinated spinal cord containing the step of administering bone marrow-derived mesenchymal stem cells into the injured spinal cord of a subject and a cell composition for neuronal regeneration of the demyelinated spinal cord.
  • the applicable subject herein is a vertebrate and preferably a mammal, more preferably any mammals except humans, preferably experimental animal models, such as rat, rabbit, guinea pig, hamster, dog, cat and more preferably apes, such as chimpanzee and gorilla.
  • the applicable subject for the method of neuronal regeneration of the demyelinated spinal cord and the possible recipient of the cell composition for neuronal regeneration of the invention can be a vertebrate and preferably a mammal.
  • the method and the composition of the present invention can be effectively used for the treatment of neurodegenerative diseases.
  • the applicable neurodegenerative diseases can be any diseases caused by abnormal destruction of neuronal cells, and the method and composition of the invention can also be effective for the treatment of motor disturbance caused by the spinal cord injury.
  • the spinal cord was extracted from an anesthetized mouse, which was then cut using a chopper to prepare the spinal cord slices in a precise thickness.
  • the spinal cord slices were cultured and treated with lysolecithin to induce demyelination. After inducing demyelination, the medium containing lysolecithin was replaced with a fresh medium.
  • Mesenchymal stem cells were injected into dorsal column of the spinal cord to induce remyelination.
  • Immunofluorescence was performed with undemyelinated group (control) , demyelinated group untreated with the stem cells and remyelinated group treated with the stem cells, followed by observation of the expression of the neuroglia cell marker protein, neuronal regeneration by histological method, and apoptosis by PI assay.
  • Fig. 1 demyelination of the spinal cord slices by lysolecithin was confirmed (see Fig. 1), and neuronal regeneration by the treatment of mesenchymal stem cells was observed (see Fig. 2) by histological method.
  • the neuronal regeneration was directional from the injured spinal cord slices to the mesenchymal stem cells, which was confirmed by measuring the increase of the neuroglia cell expression detected by immunofluorescence (see Figs. 3, 4, 5 and 6) .
  • Example 1 Transplantation of mesenchymal stem cells ⁇ !-!> Culture of human mesenchymal stem cells Human mesenchymal stem cells derived from bone marrow were purchased from Cambrex Co (USA) . These cells were sub-cultured twice in a mesenchymal stem cell growth medium (MSCGM) . Then, they were maintained in DMEM (Invitrogen, USA) supplemented with 10% FBS (Invitrogen, USA), 100 ng/ml. penicillin and 100 U/mi streptomycin (Invitrogen, USA) in a 5% CO 2 , 37 ° C incubator. ⁇ l-2> Preparation of spinal cord tissue slices
  • Sprague-Dawley rats at 16 days old were anesthetized and the spinal cord was extracted.
  • the spinal cord was put into ice-cold HBSS (Hanks balanced salt solution) .
  • Thoracic and lumbar spinal cords were cut into 400 [M in thickness by using a tissue chopper (Mcllwain tissue chopper, The Mickle Laboratory Engineering Co. LTD, England) .
  • tissue chopper Mcllwain tissue chopper, The Mickle Laboratory Engineering Co. LTD, England
  • Several spinal cord slices were loaded on 0.4 ⁇ m filter (Millicell-CM filters, Millipore, USA) placed on a 6 well plate.
  • the spinal cord slices were cultured in 1 ml of 25% horse serum (Invitrogen, USA) containing 50% MEM (Invitrogen, USA) supplemented with Earl's salts, 25% HBSS (Invitrogen, USA), 20 mM HEPES (Sigma, USA) and 6 mg/ml of D-glucose (Duchefa, Netherlands) .
  • the culture conditions were 5% CO 2 , 37 "C and the medium was replaced with a fresh medium twice a week. ⁇ l-3> Demyelination by lysolecithin
  • the spinal cord slices were cultured in vitro (DIV, days in vitro) for 7 days. After treating 0.5 mg/ml of lysolecithin, the slices were cultured at 37 ° C for 17 hours to induce demyelination. After inducing demyelination, the medium containing lysolecithin was replaced with 1 ml of fresh medium. ⁇ l-4> Cell transplantation
  • Example ⁇ 1-1> Mesenchymal stem cells of Example ⁇ 1-1> were transplanted in dorsal column of the slices prepared in

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Abstract

The present invention relates to a neuronal regeneration material screening method using ex vivo models, more precisely a neuronal regeneration material screening method using an organotypic spinal cord slice culture. The screening method for neuronal regeneration material of the present invention can be effectively used for the development of a therapeutic agent for neurodegenerative diseases.

Description

[DESCRIPTION]
[invention Title]
NEURONAL REGENERATION MATERIAL SCREENING METHOD BY EX VIVO MODEL
[Technical Field]
The present invention relates to a neuronal regeneration material screening method using ex vivo models, more precisely a screening method for a material that is able to regenerate neuronal cells by using the spinal cord tissue slices.
[Background Art]
Mesenchymal stem cells were first found in the mouse bone marrow cells, which were in the form of fibroblasts.
Mesenchymal stem cells are classified into several mesodermal series and these cells can be differentiated into various cells, that is mesenchymal stem cells are characterized by their diversity in differentiation into osteoblasts, adipocytes, endothelial cells and neuronal cells .
Understanding and interest in mesenchymal stem cells have been increased in various clinical trials and studies on the efficiency and safety of gene therapy using mesenchymal stem cells are under going. Recently, it has been reported that bone marrow-derived mesenchymal stem cells were applied to induce regeneration of bone, myocardium and nerves. In particular, this application seems to have great advantage for the treatment of various neurodegenerative diseases because the transplantation of stem cells into the wounded area not only opens the possibility of nerve replacement but also plays an important role in maintaining existing cells.
Many characteristics of in vivo spinal cord injury models, which are necessary for the study of chronic pathological phenomena caused by short-term lesion of spinal cord nerves, have been well identified. From the observation on the area neighboring damaged spinal cord was confirmed that direct damage or tissue destruction was reduced as farther from the damaged area and spinal cord injury was associated with the loss of neurons and glia. Once the spinal cord is damaged, changes in expression of various genes and proteins are observed, which finally cause demyelination, according to recent reports. In the experiment of the spinal cord injury by using in vivo system, the interpretation of the test results might be difficult because of the complicity of the in vivo system. Thus, in vitro system is more prefered, which enables precise regulation and repeated experiments with reducing costs. However, in vitro model also has a disadvantage of having no biological activity. Therefore, ex vivo model is now in the center of our concern as an alternative experimental model overcoming disadvantages of both in vivo and in vitro models. The ex vivo model such as organotypic slice cultures has been used for the study of spinal cord lesion model such as ischemia and cytotoxicity (Krassioukov et al., J Neurotrauma 19:1521-1529, 2002). The spinal cord slice culture has dorsal horn and ventral horn, distinguished from each other, and keeps its excellent form as a whole, making the confirmation of neuron distribution easy.
Demyelination of axon without structural damage and the disorder of saltatory conduction might be the cause of functional defect by spinal cord injury. Lysolecithin is a lipid containing detergent-like protein and has a similar activity with a membrane soluble agent exhibiting myelinated cell-specific toxicity to cause demyelination. That is, lysolecithin inserted in myelinated fiber such as the spinal cord in vivo causes demyelination, leading to the spinal cord injury. In the meantime, loss of oligodendrocytes, along with multiple scierosis, has been known to be a cause of various adult demyelination associated diseases, confirming the association of oligodendrocytes and myelination of the spinal cord. When the precursor cells of human oligodendrocytes were transplanted into the adult mouse brain damaged by lysolecithin, the brain cells were fast developed like oligodendrocytes and the exposed axons were myelinated even though not as firm as oligodendrocytes and with comparatively low efficiency (Goldman, Nat Biotechnol 23:862-871. 2005). These results suggest that axons demyelinated by lysolecithin can be remyelinated by the transplantation of mesenchymal stem cells.
The present inventors confirmed that neuronal regeneration by remyelination of damaged axons in spinal cord slices could be induced after transplantation of bone marrow-derived mesenchymal stem cells into those slices which had previously been demyelinated by a toxic compound. Furthermore, the present inventors completed this invention by confirming that directional neuronal regeneration could be effectively applied as a novel treatment method for neurodegenerative diseases.
[Disclosure] [Technical Problem]
It is an object of the present invention to provide a use of the directional neuronal regeneration resulted from the transplantation of mesenchymal stem cells into the spinal cord section demyelinated by a toxic compound for the treatment of neurodegenerative diseases and a use of a model established thereby for the screening of a novel drug capable of regenerating neurons.
[Technical Solution] To achieve the above object, the present invention provides a screening method for a neuronal regeneration material by using spinal cord slices.
The present invention also provides a method for neuronal regeneration of the demyelinated spinal cord containing the step of administering bone marrow-derived mesenchymal stem cells into the injured spinal cord of a subject .
The present invention further provides a cell composition for neuronal regeneration of the demyelinated spinal cord comprising bone marrow-derived mesenchymal stem cells.
[Advantageous Effect]
The method for neuronal regeneration in the demyelinated area by transplanting bone marrow-derived mesenchymal stem cells and the method for screening a neuronal regeneration material using ex vivo models of spinal cord live tissue slices of the present invention can be effectively applied as a novel treatment method for neurodegenerative diseases. [Description of Drawings]
The application of the preferred embodiments of the present, invention is best understood with reference to the accompanying drawings, wherein:
Fig. 1 is a set of photographs showing the demyelinations of spinal cord slices by lysolecithin: a: Transmittance; b: H & E staining; and, c: Immunofluorescence using MBP antibody.
Fig. 2 is a set of photographs illustrating neuronal regeneration of the demyelinated spinal cord slices by the transplantation of mesenchymal stem cells: a: Non-demyelinated group (control group); b: Demyelinated group (Lyso+PBS) ; and, c: Remyelinated group (Lyso+MSC) .
Fig. 3 is a set of photographs illustrating the immunofluorescence of cell nuclei of neurofilaments (150 kDa) to confirm neuronal regeneration of the demyelinated spinal cord slices by the transplantation of mesenchymal stem cells: a: 1 week later; and, b: 2 weeks later. Fig. 4 is a set of photographs illustrating the immunofluorescence of neurofilaments (150 kDa) to confirm neuronal regeneration of the dernyelinated spinal cord slices by the transplantation of mesenchymal stem cells: a: 1 week later; and, b: 2 weeks later.
Fig. 5 is a set of graphs illustrating the increase of the expression of neurofilaments (150 kDa) by neuronal regeneration of the demyelinated spinal cord section by the transplantation of mesenchymal stem cells: a: 1 week later; and, b: 2 weeks later.
Fig. 6 is a set of photographs illustrating the immunofluorescence using Pl and CNPase to confirm neuronal regeneration of the demyelinated spinal cord slices by the transplantation of mesenchymal stem cells: a: 1 week later; and, b: 2 weeks later.
[Mode for Invention]
Hereinafter, the present invention is described in detail .
The present invention provides a screening method for a neuronal regeneration material comprising the following steps: 1) Culturing tissue slices obtained from the spinal cord;
2) Damaging the tissue slices in course of the culture, of step 1) by treating with a toxic compound; 3) Administering neuronal regeneration material candidates to the damaged tissue slices; and
4) Selecting neuronal regeneration material candidates that exhibit significant neuronal regeneration effect, compared with the negative control untreated with such candidates.
The experiments designed with animal are classified as the in vivo model targeting an individual animal, the in vitro model targeting a cell culture and the ex vivo model targeting a live tissue. The disadvantage of in vivo model is that the regulation of various endogenous factors is difficult and thereby the result can be misinterpreted. In the meantime, the in vitro model has also disadvantages of not-guaranteeing the activity of biomaterials . The live tissue slices obtained from spinal cord of the invention can be cultured for 3 weeks since their separation is carried out promptly by using a tissue chopper. Thus, they can be used as an ex vivo system with advantages from both in vivo and in vitro systems. The system established in the present invention facilitates the study of acute and sub-acute pathophysiology in addition to enabling the regulation of associated environments with the goal of the experiment. This system has excellent reproducibility, compared with other animal models, and is economical.
The tissue slices of the present invention can survive approximately three weeks under the proper culture condition. Thus, they can be effectively applied for screening of novel materials involved in the regeneration of damaged spinal cord tissues. More particularly, after damaging neurons of the tissue slices artificially, a candidate molecule expected to induce the regeneration of the damaged neuron can be adminstered and the slices can be cultured for 2 - 3 weeks. Then, the regeneration level and characteristics can be observed to investigate the usability of the novel candidate material. In step 1), the thickness of the tissue slices is 300 μm - 500 μm, more preferably 400 μm. In step 2), the toxic compound is any substance capable of inducing demyelination of the spinal cord axon, and preferably lysolecithin. In step 4), the neuronal regeneration effect is confirmed by measuring the expression of a neuroglia cell marker protein labeled by immunofluorescence, observing neuronal regeneration by histological method, and measuring apoptotic cells by TUNEL assay.
In step 2) , the injury includes every neuronal injury in the spinal cord and preferably the injury caused by demyeli nation. In step 3), the candidate for neuronal regeneration is selected from the group consisting of compounds, growth factors, cytokines, mesenchymal stem cell originated factors, extracellular matrix proteins, proteins involved in the axonal outgrowth and factors involved in intracellular signal transduction.
The present invention also provides a method for neuronal regeneration of the demyelinated spinal cord containing the step of administering bone marrow-derived mesenchymal stem cells into the injured spinal cord of a subject and a cell composition for neuronal regeneration of the demyelinated spinal cord.
The applicable subject herein is a vertebrate and preferably a mammal, more preferably any mammals except humans, preferably experimental animal models, such as rat, rabbit, guinea pig, hamster, dog, cat and more preferably apes, such as chimpanzee and gorilla. The applicable subject for the method of neuronal regeneration of the demyelinated spinal cord and the possible recipient of the cell composition for neuronal regeneration of the invention can be a vertebrate and preferably a mammal. The method and the composition of the present invention can be effectively used for the treatment of neurodegenerative diseases. The applicable neurodegenerative diseases can be any diseases caused by abnormal destruction of neuronal cells, and the method and composition of the invention can also be effective for the treatment of motor disturbance caused by the spinal cord injury. In a preferred embodiment of the present invention, the spinal cord was extracted from an anesthetized mouse, which was then cut using a chopper to prepare the spinal cord slices in a precise thickness. The spinal cord slices were cultured and treated with lysolecithin to induce demyelination. After inducing demyelination, the medium containing lysolecithin was replaced with a fresh medium. Mesenchymal stem cells were injected into dorsal column of the spinal cord to induce remyelination. Immunofluorescence was performed with undemyelinated group (control) , demyelinated group untreated with the stem cells and remyelinated group treated with the stem cells, followed by observation of the expression of the neuroglia cell marker protein, neuronal regeneration by histological method, and apoptosis by PI assay. As a result, demyelination of the spinal cord slices by lysolecithin was confirmed (see Fig. 1), and neuronal regeneration by the treatment of mesenchymal stem cells was observed (see Fig. 2) by histological method. In particular, the neuronal regeneration was directional from the injured spinal cord slices to the mesenchymal stem cells, which was confirmed by measuring the increase of the neuroglia cell expression detected by immunofluorescence (see Figs. 3, 4, 5 and 6) .
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.
However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.
Example 1: Transplantation of mesenchymal stem cells <!-!> Culture of human mesenchymal stem cells Human mesenchymal stem cells derived from bone marrow were purchased from Cambrex Co (USA) . These cells were sub-cultured twice in a mesenchymal stem cell growth medium (MSCGM) . Then, they were maintained in DMEM (Invitrogen, USA) supplemented with 10% FBS (Invitrogen, USA), 100 ng/ml. penicillin and 100 U/mi streptomycin (Invitrogen, USA) in a 5% CO2, 37 °C incubator. <l-2> Preparation of spinal cord tissue slices
Sprague-Dawley rats at 16 days old were anesthetized and the spinal cord was extracted. The spinal cord was put into ice-cold HBSS (Hanks balanced salt solution) . Thoracic and lumbar spinal cords were cut into 400 [M in thickness by using a tissue chopper (Mcllwain tissue chopper, The Mickle Laboratory Engineering Co. LTD, England) . Several spinal cord slices were loaded on 0.4 βm filter (Millicell-CM filters, Millipore, USA) placed on a 6 well plate. The spinal cord slices were cultured in 1 ml of 25% horse serum (Invitrogen, USA) containing 50% MEM (Invitrogen, USA) supplemented with Earl's salts, 25% HBSS (Invitrogen, USA), 20 mM HEPES (Sigma, USA) and 6 mg/ml of D-glucose (Duchefa, Netherlands) . The culture conditions were 5% CO2, 37 "C and the medium was replaced with a fresh medium twice a week. <l-3> Demyelination by lysolecithin
The spinal cord slices were cultured in vitro (DIV, days in vitro) for 7 days. After treating 0.5 mg/ml of lysolecithin, the slices were cultured at 37 °C for 17 hours to induce demyelination. After inducing demyelination, the medium containing lysolecithin was replaced with 1 ml of fresh medium. <l-4> Cell transplantation
Mesenchymal stem cells of Example <1-1> were transplanted in dorsal column of the slices prepared in
Example <l-2> by using an Eppendorf Cell Tram Injector
(Eppendorf, Germany) . Example 2: Confirmation of neuronal regeneration <2-l> lmmunofluorescent staining
Spinal cord slices were fixed in 4% formaldehyde overnight at 4°C and then permeated with 0.5% Triton X-IOO for 10 minutes, followed by blocking with 3% BSA containing 0.1% Triton XlOO for one hour. Then, the slices were treated with primary antibodies such as polyclonal anti-MBP antibody (myelin basic protein; Chemicon, USA) , polyclonal anti-NF-M antibody (Neurofilament M; Chemicon, USA) , monoclonal anti-Hu antibody (Human Nuclei; Chemicon, USA) , Pl (propidium iodide, Sigma, USA) or monoclonal anti-CNPase antibody (cyclin nucleotide phosphodiesterase; Covance, USA) and then further cultured overnight at 4 °C . Then, they were incubated with the secondary antibodies such as goat anti-mouse Alexa488 (green, molecular probe, USA) or goat anti-rabbit Cy3 (red, Jackson Lab, USA) .
As a result, the expression of MBP was reduced by lysolecithin in the spinal cord slices, compared with the control (Fig. Ic). The immunofluorescence assay which was performed to examine the expression of NF-M also confirmed that directional neuronal regeneration was induced from spinal cord slices to mesenchymal stem cells (Figs. 3) . In addition, the expression of a neuroglia cell marker (CNPase) was increased (Fig. 4 and Fig. 5). PI labeled apoptotic cells were reduced in the group treated with mesenchymal stem cells, confirming neuronal regeneration by mesenchymal stem cells (Fig. 6). <2-2> Histological analysis
Spinal cord tissue slices were fixed in 10% formalin for one hour. To detect myelin, the tissue slices were stained with 0.1% Luxol fast blue. To distinguish neurons, the slices were counter-stained with H&E.
In result, serious demyelination was observed in spinal cord slices treated with lysolecithin, compared with the control (Fig. Ib), and the neuroal regeneration was also observed by the transplantation of mesenchymal stem cells (Fig. 2) .
Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

[CLAIMS]
[Claim l]
A screening method for a neuronal regeneration material comprising the following steps: 1) Culturing tissue slices obtained from the spinal cord;
2) Damaging the tissue slices in course of the culture of step 1) by treating with a toxic compound;
3) Administering neuronal regeneration material candidates to the damaged tissue slices; and
4) Selecting neuronal regeneration material candidates that exhibit significant neuronal regeneration effect, compared with the negative control untreated with such candidates.
[Claim 2]
The screening method according to claim 1, wherein the damage of step 2) is caused by demyelination.
[Claim 3]
The screening method according to claim 1, wherein the neuronal regeneration material candidate includes a compound, a growth factor, a cytokine, a mesenchymal stem cell originated factors, an extracellular matrix protein, proteins involved in the axonal outgrowth and factors involved in intracellular signal transduction.
[Claim 4] The screening method according to claim 1, wherein the tissue slices are live tissue slices cut by a chopper into slices in precise thickness.
[Claim 5] The screening method according to claim 4, wherein the live tissue slices are 300 ~ 500 μm in thickness.
[Claim β]
The screening method according to claim 1, wherein the toxic compound of step 2) is lysolecithin which induces demyelination of axons in the spinal cord.
[Claim 7)
The screening method according to claim 1, wherein the positive control group is spinal cord slices treated with mesenchymal stem cells, whereas the negative control group is spinal cord slices untreated with mesenchymal stem cells .
[Claim 8] A method for neuronal regeneration of the demyelinated spinal cord, containing the step of administering bone marrow-derived mesenchymal stem cells into the injured spinal cord of a subject.
[Claim 9]
The method according to claim 8 , wherein the subj ect is a vertebrate .
[Claim lθ]
The method according to claim 8, wherein the subject is a mammal.
[Claim 111 A cell composition for neuronal regeneration of the demyelinated spinal cord comprising bone marrow-derived mesenchymal stem cells.
PCT/KR2007/005107 2007-01-19 2007-10-18 Neuronal regeneration material screening method by ex vivo model WO2008088118A1 (en)

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Citations (2)

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WO1996015226A1 (en) * 1994-11-14 1996-05-23 Neurospheres Holdings Ltd. Regulation of neural stem cell proliferation
WO2002000849A1 (en) * 2000-06-26 2002-01-03 Renomedix Institute Inc. Cell fraction containing cells capable of differentiating into nervous system cells

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WO1996015226A1 (en) * 1994-11-14 1996-05-23 Neurospheres Holdings Ltd. Regulation of neural stem cell proliferation
WO2002000849A1 (en) * 2000-06-26 2002-01-03 Renomedix Institute Inc. Cell fraction containing cells capable of differentiating into nervous system cells

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Title
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JEFFERY N.D. ET AL.: "Remyelination of mouse spinal cord axons demyelinated by local injection of lysolecithin", J. NEUROCYTOL., vol. 24, no. 10, October 1995 (1995-10-01), pages 775 - 781 *
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