# Patent

## Title
Immortalized mesenchymal stromal cell from adipose tissue

## Abstract

The present invention relates to a composition for diagnosing a Parkinson's disease comprising mesenchymal stromal cells derived from adipose tissue, a method of providing information for diagnosing Parkinson's disease and/or the extent of the disease progression, a biomarker for diagnosing a Parkinson's disease, a method of screening a drug candidate treating Parkinson's disease where the drug candidate is a target of the biomarker, and an immortalized mesenchymal stromal cell from adipose tissue.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation of U.S. application Ser. No. 12/972,315, filed Dec. 17, 2010, which claims priority to Korean Patent Application No. 10-2010-0058273, filed Jun. 18, 2010, the entire contents of which are incorporated herein by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a composition for diagnosing a Parkinson's disease comprising mesenchymal stromal cells derived from adipose tissue, a method of providing information for diagnosing Parkinson's disease and/or extent of disease progression using mesenchymal stromal cells derived from adipose tissue, a biomarker for diagnosing a Parkinson's disease, aa method of screening an agent treating Parkinson's disease where the agent targets the biomarker, and an immortalized mesenchymal stromal cell from adipose tissue.
2. Description of the Related Art
Parkinson's disease (PD) is a secondarily common neurodegenerative disease which about one percent of old people aged over 60 suffers from, but the cause of disease has not yet been determined.
It has been suggested that Parkinson's disease has some connections with the selective loss of dopaminergic neurons in substantia nigra, and with the extensive neuron changes causing various complex motile and immotile symptoms.
A genetic mutation of disease-causing gene such as α-synuclein, parkin, Parkinson disease autosomal recessive, early onset 7 (DJ-1), or phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1) is mentioned as a cause of familial Parkinson's disease.
Parkin acts as E3 ligase in ubiquitin-proteasomal system, protects against the oxidative stress, and helps the maintenance of mitochondrial function. The mutation in Parkin gene can cause a hereditary early-onset of Parkinson's disease.
The correlation between mitochondrial dysfunction and Parkinson's disease can be observed as the widely known disease-causing mechanism in PD patient subgroup involves aberrant shape and dysfunction of mitochondria.
The damage of mitochondrial function increases an oxidative stress and associates with the control of calcium homeostasis and cell apoptosis pathway. The oxidative stress can be defined as one of causes inducing apoptosis of dopaminergic neuronal cells of the substantia nigra in Parkinson's disease patient.
Parkinson's disease associated gene products including α-synuclein, Parkin, PINK′, DJ-1 and the like can be found in mitochondria and play a critical role in the mitochondrial dysfunction and oxidative stress.
The methods of diagnosing Parkinson's disease and determining extent of the disease progression include a method of imaging brain nigros-triatal region with a magnetic resonance image (MRI) analysis, a positron emission tomography (PET), a single photon emission computed tomography (SPECT), and the like, and a method of analyzing a sample taken from brain tissue with a biomarker. However, such methods still lead to inaccurate analysis of results, and unwanted pain and a risk to patient from directly taking a sample form brain tissue.
SUMMARY OF THE INVENTION
To solve the problems in the art, the present inventors found that Parkinson's disease could be diagnosed by using the mesenchymal stromal cell derived from adipose tissue, developed a technology for accurately diagnosing Parkinson's disease without directly taking brain tissue, and completed the present invention by developing a biomarker for diagnosing Parkinson's disease using the technology.
Therefore, an embodiment of the present invention provides a composition for diagnosing Parkinson's disease comprising the mesenchymal stromal cell derived from adipose tissue.
Another embodiment provides a method of providing an information for diagnosing Parkinson's disease and determining extent of the progression of Parkinson's disease.
Further embodiment of the present invention provides a biomarker for diagnosing Parkinson's disease where the biomarker is obtained from the mesenchymal stromal cell derived from the adipose tissue.
Still another embodiment of the present invention provides a method of screening a drug treating Parkinson's disease where the drug targets the biomarker.
Another embodiment provides an immortalized mesenchymal stromal cell from adipose tissue
DETAILED DESCRIPTION OF THE EMBODIMENTS
The present invention can provide a tool for understanding and determining the brain physiological states without isolating the brain tissue from Parkinson's disease patient, by performing the transcriptome microarray analysis of mesenchymal stromal cell derived from early-passage adipose tissue taken from human subject with early-onset (congenital) hereditary Parkinson's disease such as Parkin-deficient PD, as well as late-onset (acquired) Idiopathic Parkinson's disease (idiopathic PD).
The present inventors completed the present invention by separating a human mesenchymal stromal cell derived from human adipose tissue (hAD-MSC), from a subject with Idiopathic PD or Parkin-deficient Parkinson's disease, and comparing the gene expression pattern of the mesenchymal stromal cell with that of non-Parkinson's disease. The human adipose tissue is abundant and easily accessible source for mesenchymal stem cells (MSC).
Hereinafter, hAD-MSC obtained from a patient with idiopathic Parkinson's disease is referred to as ‘PD,’ hAD-MSC obtained from a patient with Parkin-deficient Parkinson's disease as ‘Parkin,’ and hAD-MSC obtained from a patient who has pituitary adenoma without Parkinson's disease as ‘non-PD’ or ‘PA.’ Initially, by analyzing differentially-expressed gene (DEG) among three groups, 413 genes are confirmed to be differentially expressed, and then classified into three groups of non-PD, PD and Parkin. In addition, DEG are analyzed and divided to seven clusters according to K-mean clustering analysis, and the GENBANK accession numbers are listed in Tables 6a-6e. In addition, the functional groups of human biomarker candidates are organized, and non-PD vs. PD and non-PD vs. Parkin are compared. Finally, the PD associated DEGs which are regulated differently due to the oxidative stress are classified into one of groups among non-PD, PD and Parkin categories.
The knowledge of selective gene expression pattern in Parkinson's disease patient gained from the present invention can be helpful for obtaining the physiological information and early-diagnosis of Parkinson's disease, and developing an effective target specific drug for treating the Parkinson's disease by using the genes as a biomarker.
First of all, the present invention relates to a composition for diagnosing a disease, comprising mesenchymal stromal cells derived from adipose tissue, where the disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Huntington's disease, hereditary dystonia, hereditary dyskinesia, and metabolic disease. The present inventors found that the presence of Parkinson's disease and the extent of disease progression could be diagnosed without separating the brain tissue from Parkinson's disease patient by assaying the gene expression pattern of mesenchymal stromal cell derived from adipose tissue, since the gene expression is very similar to that of brain tissue. In accordance with the present invention, the use of adipose tissue-derived mesenchymal stromal cell for diagnosing the Parkinson's disease removes the step of dangerous separation of brain tissue from the subject. The diagnosing method using the adipose tissue-derived mesenchymal stromal cells can be widely applied for diagnosing various diseases such as Alzheimer's disease, Huntington's disease, hereditary dystonia, hereditary dyskinesia, and metabolic disease.
The subject includes any kind of mammals, and preferably human beings who are suffering or are likely to suffer from Parkinson's disease, Alzheimer's disease, Huntington's disease, hereditary dystonia, hereditary dyskinesia, and metabolic disease. Since one of the common causes of the diseases is adipose tissue (Human Lipodystrophies: Genetic and Acquired Diseases of Adipose Tissue; Capeau J, et al.; 2010; PUBMED), the application of following diagnosing technology for Parkinson's disease can be extended to diseases listed above.
The adipose tissue can be one separated from mammals, preferably human. The separated region is not limited to a particular region of body and can include any regions of a body such as breast and abdominal region. For more accurate analysis, the separated adipose tissue can be used preferably after eliminating cell debris and blood cell.
Hereinafter, the term, adipose tissue can be separated or unseparated one from a live body, and includes an adipose cell.
In an embodiment of the present invention, the mesenchymal stromal cells (hAD-MSC) can be, for example, but are not limited thereto, cells showing the mononuclear cell properties which are confirmed by positive expression of human integrin beta-1 marker CD29, phagocytic glycoprotein-1 marker CD44, and human integrin alpha-4 marker CD49d, and at the same time expressing slightly primitive hematopoietic precursors and vascular endothelial marker CD34, vascular endothelial marker CD31 and vascular adhesion molecule 1, (VCAM-1) marker CD106.
The Parkinson's diseases which can be detected by using the composition of the present invention includes all kinds of the Parkinson's diseases such as an acquired Idiopathic Parkinson's disease, congenital, familial Parkinson's disease (for examples, Parkin, α-synuclein, phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1, a mitochondrial kinase), Parkinson disease autosomal recessive, early onset 7 (DJ-1), Leucine-rich repeat kinase 2 (LRRK2), and High temperature requirement protein A2 (HTRA2) deficient).
In another embodiment of the present invention, a method of diagnosing Parkinson's disease using the mesenchymal stromal cells derived from the adipose tissue is provided.
More specifically, the method comprises the steps of separating a mesenchymal stromal cell derived from adipose tissue of a subject, assaying a gene expression pattern of the mesenchymal stromal cell; and determining the presence of disease by analyzing the assayed result or the disease progression degree by analyzing the assayed result.
The assaying step of gene expression pattern of hAD-MSC can be performed by any methods of analyzing gene expression which are used commonly in the art. For examples, the assaying of gene expression can be carried out by microarray analysis, Reverse transcriptase Polymerase Chain Reaction (RT-PCR), Real time Polymerase Chain Reaction (real time PCR), genomics, proteomics, microRNA assay, SNP analysis, mitochondrial assay, functional assay and the like, but not limited thereto.
As a result of the molecular biological studies in the present invention, a high-throughput microarray analysis of hAD-MSC obtained from a patient with Idiopathic Parkinson's disease, a patient with Parkin-deficient Parkinson's disease, and a patient with non-Parkinson's disease can be established, and the analysis result is compared with that of a patient with non-Parkinson's disease (control group) to identify the gene groups which are differentially expressed between a patient with Idiopathic Parkinson's disease and/or a patient with Parkin-deficient Parkinson's disease. Thus, the identified genes in a patient with non-Parkinson's disease, a patient with Idiopathic Parkinson's disease and a patient with Parkin-deficient Parkinson's disease can contribute to the understanding of physiological symptoms of Parkinson's disease, and can provide a useful tool for developing the early-diagnosis and effective treatment of Parkinson's disease targeting the human biomarker.
The mitochondrial dysfunction and increased oxygen stress are shown in subgroup of Parkinson's disease patient, and these suggest the important effect of mitochondrial dysfunction and oxygen stress on onset of Parkinson's disease. Thus, the mitochondria can be effective target for developing a biomarker of Parkinson's disease. The biochemical methods of detecting the potential biomarker of Parkinson's disease include the gene screening method, mitochondria complex I measurement, blood level of alpha-synuclein and isoforms measurement. Gene test tools which are commercially available for detecting mitochondria mutant genes such as Parkin, PINK1, and alpha-synuclein can be used. Furthermore, Coenzyme Q10, antioxidant and electron transporter (electron transporter chain component) act as an electron transporter for mitochondria complex I and II.
The genes which show differential expression at least two times in non-PD patient vs. Parkin, and non-PD vs. PD, and Parkin vs. PD are identified with hierarchical clustering analysis (FIG. 4), and summarized in table 4 to select the PD-related genes (SCUBE3, IL8, ATP1B1, TNFRSF11B, FABP3, CXCL1). IL8 and CXCL1 are chemokines which act a basic role in development, homeostasis and immune systems, and involve in inflammation of cranial nerve of Parkinson's disease patient. SCUBE3 accompanies an important molecule in dopaminergic neuron of ventral midbrain, and TNFRSF11B involves in an inflammation in neurodegeneration of Parkinson's disease. The single heterozygous mutation of ATP is suggested to be related with a cause of early-onset of Parkinson's disease. Finally, FABP3 has been used as a diagnostic marker for Parkinson's disease. The PD-related genes in non-PD vs. Parkin and Parkin vs. PD are summarized in Table 5 (SYT14, LGR5, TGFB3, ITGA2, F2RL2, DRD1, PENK, GNA14, EDNRB, HSPA2, SLC6A6, AKR1B1, and PRG4). SYT14 is a transmembrane protein involved with control of membrane trafficking. LGR5, F2RL2, DRD1, GNA14 and EDNRB involve in a signal pathway of G-protein, TGFB3 involves in a susceptibility of Parkinson's disease patient, and ITGA2 in a neuronal adhesion. An increased expression of PENK can be a cause of treatment-related dyskinesia in Parkinson's disease patient. Parkin, as a substrate for parkin, mediates the ubiquitination of HSPA2 and a molecular chaperone. SLC6A6, also known as taurine, is a neurotransmitter. Taurine is a beta-amino acid abundantly located in substantia nigra (SN), and functions as a neurotransmitter in substantia. The immunoreactivity of AKR1B1 is generated in human cerebral cortex and hippocampus, and PRG4 relates with the inclusions of Parkinson's disease.
The comparison analysis of seven regulating sequences of K-mean clustering genes in non-PD, PD and Parkin are shown in FIG. 5b. The names and GENBANK accession numbers of PD-related genes showing the increased or decreased gene expression are summarized in Tables 6a-6e. The genes showing the increased gene expression pattern are shown in Table 6a (ITGA8, CTSH, CCRL1), Table 6b (TGFB3, DRD1, GNA14, PENK, PRG4, LGR5, HLA-DPA1), and Table 6e (SCUBE3, HSPA2, TGFB3, DRD1, GNA14, PENK, PRG4, LGR5, RELN, EDNRB, ITGA2, SLC6A6, F2RL2, CDK6, AKR1B1, MMP8, ID1, NEFM, ATP1B1, TNFRSF11B, TNFRSF10D), and the genes showing the decreased gene expression pattern are shown in Table 6c (BEX1) and Table 6d (IL8, CXCL6). The change in gene expression seems to be due to the mitochondria activity change between late-onset of Idiopathic Parkinson's disease and congenital early-onset of Parkinson's disease or due to compensation therebetween. These data also suggest the potential biomarker for the onset of Parkinson's disease. The molecular functional groups in non-PD vs. PD and non-PD vs. Parkin are determined and shown in FIG. 8a to FIG. 8d, and the genes which show the change in the gene expression level in Idiopathic Parkinson's disease patient and Parkin-deficient Parkinson's disease patient can provide a potential human biomarker candidate for detecting a disease onset and a selective vulnerability.
In addition, after analyzing of genes differentially expressed due to the oxygen stress in PD, the genes and the clusters taken from Cluster Nos. 2 to 6 are shown in Table 9. Interestingly, genes showing a linear increase of gene expression were discovered in all groups, which may be explained by an increased compensation against vulnerability caused by oxygen stress in pathology of Idiopathic PD and Parkin-deficient PD.
Based on these results, when there is an increased expression of at least one selected from the group consisting of ITGA8, CTSH, CCRL1, TGFB3, DRD1, GNA14, PENK, PRG4, LGR5, HLA-DPA1, SCUBE3, HSPA2, RELN, EDNRB, ITGA2, SLC6A6, F2RL2, CDK6, AKR1B1, MMP8, ID1, NEFM, ATP1B1, TNFRSF11B, and TNFRSF10D in mesenchymal stromal cells derived from adipose cell of a patent, or when where is an decreased expression of at least one selected from the group consisting of BEX1, IL8, and CXCL6 in mesenchymal stromal cells derived from adipose cell of a patent, it is possible to determine the presence of the Parkinson's Disease. Parkinson's disease includes late-onset (acquired) Parkinson's disease (Idiopathic Parkinson's disease), or early-onset (congenital, familial, hereditary) Parkinson's disease (for examples, Parkin, α-synuclein, phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1, a mitochondrial kinase), Parkinson disease autosomal recessive, early onset 7 (DJ-1), Leucine-rich repeat kinase 2 (LRRK2), or High temperature requirement protein A2 (HTRA2) deficient Parkinson's disease and etc).
In an embodiment of the present invention, the increase and decrease of the gene expressions can be measured by the amount of protein expressed. When the amount is about 1.5 to 3 times higher than that of normal group without Parkinson's disease, the result can be determined to be significant.
In an embodiment of the present invention, a method of screening a drug treating Parkinson's disease, in which the drug targets the biomarker in mesenchymal stromal cells derived from adipose tissue, is provided.
More specifically, the method comprises the steps of contacting a drug candidate with a mesenchymal stromal cell derived from adipose tissue; and assaying a gene expression pattern of the mesenchymal stromal cell; and determining the drug candidate as a drug treating Parkinson's disease in case that there is a difference of gene expression pattern in the mesenchymal stromal cells between the treatment and non-treatment of the drug candidate, wherein at least gene is selected from the group consisting of ITGA8, CTSH, CCRL1, TGFB3, DRD1, GNA14, PENK, PRG4, LGR5, HLA-DPA1, SCUBE3, HSPA2, RELN, EDNRB, ITGA2, SLC6A6, F2RL2, CDK6, AKR1B1, MMP8, ID1, NEFM, ATP1B1, TNFRSF11B, TNFRSF10D, BEX1, IL8, and CXCL6.
For example, when the group treated with the drug candidate shows the increased gene expression (preferably, at least an increase of two-fold) of at least one selected from the group consisting of ITGA8, CTSH, CCRL1, TGFB3, DRD1, GNA14, PENK, PRG4, LGR5, HLA-DPA1, SCUBE3, HSPA2, RELN, EDNRB, ITGA2, SLC6A6, F2RL2, CDK6, AKR1B1, MMP8, ID1, NEFM, ATP1B1, TNFRSF11B, TNFRSF10D and the like, or the decreased gene expression (preferably, at least a decrease of two-fold) of at least one selected from the group consisting of BEX1, IL8, CXCL6 and the like, compared to that of the group untreated with the drug candidate, the drug candidate can be determined as a drug for treating Parkinson's disease, for examples, late-onset (acquired) Parkinson's Disease (Idiopathic Parkinson's Disease), or early-onset (congenital, familial, hereditary) Parkinson's Disease (for examples, Parkin, α-synuclein, phosphatase and tensin homologue (PTEN)-induced putative kinase 1 (PINK1, a mitochondrial kinase), Parkinson disease autosomal recessive, early onset 7 (DJ-1), Leucine-rich repeat kinase 2 (LRRK2), or High temperature requirement protein A2 (HTRA2) deficient Parkinson's disease, etc.
The increase or decrease of gene expression can be measured by any method of measuring the gene expression level which has been known generally in the art, for examples but not limited to, microarray assay, Reverse transcriptase polymerase chain reaction (RT-PCR), Real time PCR, genomics, proteomics, microRNA assay, SNP analysis, mitochondrial assay, functional assay and the like.
The data obtained in the present invention provide a predictable scenario for onset of Parkinson's disease. In conclusion, the gene expression analysis of the mesenchymal stromal cells derived from the human adipose tissue can identify specific molecular functional groups of the genes which are affected by the mitochondrial dysfunction and oxidative stress. Thus, the present invention provides a technology for diagnosing Parkinson's disease and/or determining its extent of disease progression using the mesenchymal stromal cell derived from adipose tissue instead of brain tissue, for. The genes which show the change in gene expression in Idiopathic or Parkin-deficient (familial) Parkinson's disease patient compared to the non-Parkinson's disease can be identified by using such technology, and can be used both as a biomarker as well as a target for developing a drug treating Parkinson's disease.



BRIEF DESCRIPTION OF DRAWINGS
FIGS. 1a and 1b show the procedure of separating and culturing the mesenchymal stromal cells derived from adipose tissue of human patient with Parkinson's disease.
FIG. 2 represents schematically the procedure of preparing a stock by culturing the mesenchymal stromal cells derived from adipose tissue of human patient with Parkinson's disease.
FIG. 3 is a picture showing the transient change in cell morphology of the mesenchymal stromal cells derived from adipose tissue of human patient with Parkinson's disease based on culture time variations.
FIG. 4 is a Venn diagram of Differentially Expressed Gene (DEG) between the control group (non-PD, PA) and test group with Parkinson's disease.
FIG. 5a is a result of hierarchical clustering analysis of the genes showing the gene expression level at least two times higher between non-PD, PD and Parkin groups.
FIG. 5b shows the seven clusters obtained by reorganizing the expression pattern graphs which are classified through the K-mean clustering analysis of non-PD, PD and Parkin.
FIG. 6 represents the clustering analysis result of the genes showing linearly-decreased gene expression between non-PD, PD, and Parkin groups.
FIG. 7 represents the clustering analysis result of the genes showing linearly-increased gene expression between non-PD, PD, and Parkin groups.
FIG. 8a to 8d show the result classifying human biomarker which are obtained by reprogramming the molecular functional groups by using Gene Ontology and Panther database system between non-PD vs. PD and non-PD vs. Parkin.
FIG. 9 shows the comparison of cell morphology before and after immortalization with hTERT in non-PD, PD, and Parkin group.
After: the cell morphology shortly after immortalization with hTERT.
6 months: the cell morphology after immortalization with hTERT and culturing for 6 months.
1 year: the cell morphology after immortalization with hTERT and culturing for 1 year.
FIG. 10 shows a result of chromosomal structure (karyotype) analysis.
after: the karyotype analysis result shortly after immortalization with hTERT.
1 year: the karyotype analysis result after immortalization with hTERT and culturing for 1 year.
FIG. 11a shows a result of biochemical enzyme assay of mitochondria respiration chain of immortalized cell, and FIG. 11b is the result of western blot analysis showing successful separation of mitochondria in the assay of FIG. 11a. 
FIG. 12 is electronic microscopic images of mitochondria in early-culture stage and immortalization-culture stage of the cells (a, b: mitochondria in Non-PD; c, d: mitochondria in PD; and e, f: mitochondria in Parkin).
FIG. 13a represents a result of western blot analysis (left side) which shows the change in the gene expression of mitochondrial markers in early-culture stage of non-PD, PD, and Parkin groups, and a graph (right side) showing the quantitative analysis of the result.
FIG. 13b represents the change in the gene expression of HSP60 as mitochondria marker in immortalization-culture stage of the cells in non-PD, PD, and Parkin (left side) and a graph (right side) showing the quantitative analysis of the result.
FIG. 13c represents the change in the gene expression of HSP90 as mitochondria marker in immortalization-culture stage of the cells in non-PD, PD, and Parkin (left side) and a graph (right side) showing the quantitative analysis of the result.
FIG. 14a represents a western blot analysis showing the change in the gene expression of autophagy makers in immortalization-culture stage of the cells in non-PD, PD, and Parkin.
FIG. 14b represents a western blot analysis showing the change in the gene expression of mTOR as an autophagy maker in immortalization-culture stage of the cells in non-PD, PD, and Parkin of FIG. 14a. 
FIG. 14c represents a western blot analysis showing the change in the gene expression of S6K as an autophagy maker in immortalization-culture stage of the cells in non-PD, PD, and Parkin of FIG. 14a. 
FIG. 15a represents a western blot analysis showing the change in the gene expression of autophagy markers in immortalization-culture stage of the cells in non-PD, PD, and Parkin, and FIG. 15b is a graph showing the quantitative analysis of the result.
FIG. 16a represents western blot analysis showing the change in the gene expression of autophagy markers in immortalization-culture stage of the cells in non-PD, PD, and Parkin.
FIG. 16b is a graph showing the quantitative analysis of the change in the gene expression of autophagy markers in immortalization-culture stage of the cells in non-PD, PD, and Parkin.
FIG. 17 shows an analysis result of properties of the mesenchymal stromal cells derived from the adipose tissue.



EXAMPLE
The present invention is further explained in more detail with reference to the following examples. These examples, however, should not be interpreted as limiting the scope of the present invention in any manner.
Example 1
Preparation of the Mesenchymal Stromal Cell Derived from the Adipose Tissue
1.1. Separation and Culture of the Mesenchymal Stromal Cells Derived from the Adipose Tissue
The adipose tissue-derived mesenchymal stromal cells (hAD-MSC) were separated from Idiopathic Parkinson's disease (Idiopathic PD) patient, Parkin-deficient Parkinson's disease (Parkin-deficient PD) patient, and a pituitary adenoma patient who did not suffer from Parkinson's disease, and then subsequently were cultured. Hereinafter, otherwise particularly defined, hAD-MSC obtained from Idiopathic PD patient is referred to as “PD,” hAD-MSC obtained from Parkin-deficient Parkinson's disease patient to as “Parkin”, and hAD-MSC obtained from Parkinson's disease to as “non-PD” or “PA.”
These tests were performed under the permission of Institutional Review Board of Seoul National University Hospital (IRB No. 0707-024-212) and the written consent of the patients. During the performance of Deep Brain Stimulation (DBS) surgery of Idiopathic PD patient, and Parkin-deficient PD patient, the adipose tissue under the skin of clavicle was taken in order to compare with that of a pituitary adenoma patient (control group) who did not suffer from Parkinson's disease.
The adipose tissue was added to 1% antibiotic/antimyotic (Gibco®Invitrogen, Carlsbad, Calif.) in sterilized PBS (phosphate-buffered saline, pH7.4) and transferred to test room. The adipose tissue was washed with PBS three times to remove tissue debris and red blood cell, and then finely cut into small pieces. The adipose tissue was digested with 0.075% collagenase Type I (Sigma-Aldrich, St. Louis, Mo., USA) at 37° C. for 1 hour, inactivated with the same volume of DMEM/10% Fetal Bovine Serum (FBS) (Gibco®Invitrogen, Carlsbad, Calif.), and centrifuged at 1200×g for 10 minutes. The obtained pellet was cultured in three times volume of red blood lysis buffer (QIAGEN, valencia, CA, USA) at 37° C. for 10 minutes, and filtered with 100 μm strainer. The filtrate was centrifuged at 1200×g for 10 minutes. The resultant pellet was washed with PBS and centrifuged at 1200×g for 10 minutes. Finally, the obtained pellet was resuspended in Mesenchymal Stem cell Expansion medium (Millipore, SCM015, Billerica, Mass., USA) and placed onto 25T culture flask. After the cells were cultured in Mesenchymal Stem cell Expansion medium (Millipore, SCM015, Billerica, Mass., USA) at 37° C. for 48 hours, the cells were washed with PBS and the unattached cells were removed. The culture medium was replaced with new medium every three days.
As a result, the hAD-MSC was obtained, wherein two cells are obtained from a pituitary adenoma patient who did not suffer from Parkinson's Disease (PA1 and PA2), two cells are obtained from Idiopathic PD (PD1 and PD2), and two cells are obtained from Parkin-deficient PD (Parkin1 and Parkin2).
The culture procedure is shown schematically in FIGS. 1a and 1b. FIG. 2 represents schematically the procedure of preparing stock by culturing mesenchymal stromal cells derived from adipose tissue of human patient with Parkinson's disease. FIG. 3 is a picture showing the transient change in cell morphology of mesenchymal stromal cells derived from adipose tissue of human patient with Parkinson's disease according to the culture time variations. In FIG. 3, the pictures of cell morphology of adipose tissue-derived mesenchymal stromal cells were taken every day during continuous culture.
The information on the cell culture of mesenchymal stromal cells derived from adipose tissue obtained from Idiopathic PD patient, Parkin-deficient PD patient, and control group are summarized at Tables 1 to 3:







TABLE 1







Culture information of adipose tissue


obtained from Idiopathic PD patient









Patient No.
Labeling
Culture Date












1
FSC-PD#2
2007 Mar. 9


2
FSC-PD#3
2007 Apr. 2


3
FSC-PD#5
2007 Aug. 13


4
FSC-PD#6
2007 Oct. 22


5
FSC-PD#7
2007 Oct. 29


6
FSC-PD#8
2007 Nov. 19


7
FSC-PD#9
2008 Mar. 24


8
FSC-PD#10
2008 Jul. 7


9
FSC-PD#11
2008 Aug. 29


10
FSC0714
2008 Jul. 14


11
FSC0721
2008 Jul. 21


12
FSC0829
2008 Aug. 29


13
FSC1006
2008 Oct. 6


14
FSC0119
2009 Jan. 19


15
FSC0209
2009 Feb. 9


16
FSC0420
2009 Apr. 20


17
FSC0427
2009 Apr. 27


18
FSC0601
2009 Jun. 1


19
FSC0622
2009 Jun. 22


20
FSC0918
2009 Sep. 18


21
FSC1123
2009 Nov. 23
















TABLE 2







Culture information of adipose tissue obtained


from Parkin-deficient PD patient









Patient
Labeling
Culture Date





1
FSC-parkin
2007 May 17


2
gFSC
2008 Jun. 2
















TABLE 3







Culture information of adipose tissue obtained from control group









Patient No.
Labeling
Culture Date












1
FSC-#1
2006 Nov. 22


2
FSC-#2
2006 Nov. 23


3
FSC-#3
2006 Dec. 18


4
FSC-#4
2006 Dec. 18


5
FSC-#7
2007 Jan. 3


6
FSC-#8
2007 Jan. 22


7
FSC-#9
2007 Jan. 31


8
FSC-#11
2007 Feb. 15


9
FSC-#12
2007 Feb. 26


10
FSC-#14
2007 Mar. 15


11
FSC-#15
2007 Apr. 20


12
FSC-#17
2007 Oct. 2


13
FSC-#18
2007 Oct. 4


14
FSC-#19
2008 Mar. 25


15
FSC1013
2008 Oct. 13


16
FSC1014
2008 Oct. 14


17
FSC1103
2008 Nov. 3


18
FSC1104
2008 Nov. 4


19
FSC0629
2009 Jun. 29


20
FSC0630
2009 Jun. 30


21
FSC0706
2009 Jul. 6


22
FSC1019
2009 Oct. 19


23
FSC1102
2009 Nov. 2


24
FSC1109
2009 Nov. 9


25
FSC1201
2009 Dec. 1









1.2. Fluorescence-Activated Cell Sorter (FACS) Analysis

The hAD-MSC culture was separated with PBS and subsequently cultured with following primary antibodies (culture medium: Mesenchymal Stem cell Expansion medium (Millipore, SCM015, Billerica, Mass., USA), culture temperature: 37° C.).
Primary Antibody:
Anti-CD29, anti-CD44, anti-CD34, anti-CD31 (DakoCytomation, Carpinteria, Calif., USA), anti- and CD49d, anti-CD106 (Chemicon, Temecula, Calif., USA).
The cells were cultured on ice for 30 minutes, and then washed with 0.5% BSA and 2 mM EDTA in BSA (Sigma-Aldrich, St. Louis, Mo., USA). The morphological characteristics of hAD-MSC and quantitative analysis were performed by FACS SCAN flow cytometer (Becton Dickinson, San Diego, Calif., USA) and CellQuest software (Becton Dickinson, San Diego, Calif., USA).
The results are shown in FIG. 17. hAD-MSC obtained from idiopathic PD patient, Parkin-deficient PD patient, and control group were separated and cultured. Then, the cells showed the characteristics of mononuclear cell based on the expression of human integrin beta-1 marker CD29, phagocytic glycoprotein-1 marker CD44, and human integrin alpha-4 marker CD49d. In addition, the cells expressed slightly primitive hematopoietic precursors and vascular endothelial marker CD34, vascular endothelial marker CD31 and vascular adhesion molecule 1, (VCAM-1) marker CD106.
1.3. Preparation of RNA Sample
According to manufacturer's manual, the RNA sample was prepared. Specifically, whole RNA was separated with RNEASY Mini Kit columns (Qiagen, Hilden, Germany) according to the manufacturer's manual. The quantity of RNA was assessed with Agilent 2100 bioanalyser using RNA 6000 NANO CHIP (Agilent Technologies, Amstelveen, The Netherlands) and determined with ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).
1.4. Analysis of hAD-MSC Properties
hAD-MSC was separated from Idiopathic PD patient, Parkin-deficient PD patient, and the control group and cultured. In flow cytometry analysis, at least 95% of MSC (≧95%) expressed CD105, CD73 and CD90, and the cells were deficient in the expression of CD45, CD34, CD14 or CD11b; CD79□ or CD19; and HLA class II (positive at most 2%) (M Dominici, K Le Blanc, I Mueller, I Slaper-Cortenbach, F Marini, D Krause, et al, Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement, Cytotherapy, 8, 315-7, 2006). The accession numbers of the genes are summarized in following table:













Gene name
Gene accession number








CD105
NM_001114753



CD73
NM_002526



CD90
NM_006288 NM_033209



CD45
NM_002838



CD34
NM_001025109



CD14
NM_000591



CD11b
NM_000632



CD79
NM_001783



CD19
NM_001178098



HLA class II
NM_000449









The cells should be able to differentiate into osteoblasts cell, adipose cell (adipocytes) and chondroblasts under the standard in vitro differentiation condition (M Dominici, K Le Blanc, I Mueller, I Slaper-Cortenbach, F Marini, D Krause, et al, Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement, Cytotherapy, 8, 315-7, 2006).
The separated hAD-MSC were cultured at Mesenchymal Stem cell Expansion medium (Millipore, SCM015, Billerica, Mass., USA) at 37° C., and the expression profile of human integrin beta-1 marker CD29, phagocytic glycoprotein-1 marker CD44, and human integrin alpha-4 marker CD49d were analyzed with FACS analysis method. The result is shown in FIG. 17. CON in FIG. 17 shows FACS analysis result of mesenchymal stromal cells themselves.
The accession numbers of the genes utilized are summarized in following table:













Gene name
Gene accession number








CD29
NM_002211



CD44
NM_000610



CD49d
NM_000885



CD34
NM_001025109



CD31
NM_000558



CD106
NM_001078









The positive expression of the genes confirmed that the cells showed the properties of the mononuclear cell. The cells expressed a small amount of primitive hematopoietic precursors and vascular endothelial marker CD34, vascular endothelial marker CD31 and vascular adhesion molecule 1, (VCAM-1) marker CD106.
Example 2
Gene Profiling of Adipose Tissue-Derived Mesenchymal Stromal Cell
2.1. cDNA Microarray Analysis
The gene expression analysis was performed with Affymetrix GeneChip® Human Gene 1.0 ST oligonucleotide array (DNA LINK, INC (Seoul, Korea)).
According to AFFYMETRIX manufactor's protocol (www.affymetrix.com), 300 ng of RNA was added to each sample. Namely, 300 ng of all RNA per a sample was changed into double stranded cDNA. The double stranded cDNA was obtained by using SUPERSCRIPT II Reverse Transcriptase, DNA polymerase I and random hexamer inserted by T7 promoter (AFFYMETRIX GENECHIP® WT cDNA Synthesis and Amplipicaition Kit, Cat No. 900672). An amplified RNA (cDNA) was produced by in vitro transcription (IVT) with IVT Enzyme Mix (AFFYMETRIX GENECHIP® WT cDNA Synthesis and Amplipicaition Kit, Cat No. 900672), and separated with AFFYMETRIX sample cleanup module. The amplified cRNA was mixed with IVT cRNA binding buffer and 100% EtOH, and bonded in cRNA cleanup spin column. The column was washed with cRNA wash buffer, and then eluted with RNase-free water.
cDNA was reproduced with dNTP mixture including dUTP AFFYMETRIX GENECHIP® WT cDNA Synthesis and Amplipicaition Kit, Cat No. 900672), according to the random-primed reverse transcription. Then, the produced cDNA was fragmented by using UDG and APE 1 restriction enzyme (AFFYMETRIX GENECHIP® WT Terminal Labeling Kit, Cat No. 900670), and the end labeling was performed by inserting biotinylated dideoxynucleotide with TdT (Terminal deoxynucleotidyl transferase) enzyme.
According to Gene Chip Whole Transcript (WT) Sense Target Labeling Assay Manual (AFFYMETRIX), the end-labeled and fragmented cDNA was hybridized at 60 r/min, at 45° C., for 16 hours with GENECHIP® Human Gene 1.0 ST array. Then, the array was stained and washed in GENECHIP Fluidics Station 450 (AFFYMETRIX), and scanned with GENECHIP Array scanner 3000 7G (AFFYMETRIX).
2.2. Classification of DEG Included in Three Groups of Non-PD (Control Group), Idiopathic PD and Parkin-Deficient PD
To identify DEG based on two-fold gene expression difference between non-PD vs. Parkin, non-PD vs. PD and Parkin vs. PD, hierarchical clustering analysis (Eisen M B, Spellman P T, Brown P O, Botstein D, 1998) Cluster analysis and display of genome-wide expression patterns. Genetics Vol. 95, Issue 25, 14863-14868) was performed. The hierarchical clustering analysis is data-mining algorithm used for defining similarity or dissimilarity of expressed genes. By using all genes showing the gene expression difference of two-fold between three groups of non-PD, PD, Parkin as a standard, the hierarchical clustering analysis was carried out to identify the gene having high similarity and the Euclidean distance was used as a similarity measurement.
The obtain result is shown as a Venn diagram in FIG. 4. In order to identify genes showing expression level difference of at least two-fold between the groups of non-PD, PD and Parkin, after selecting genes showing expression level difference of at least two-fold in each non-PD vs Parkin, non-PD vs PD, and Parkin vs PD, and comparing with the results among non-PD vs Parkin, non-PD vs PD, and Parkin vs PD, the genes that showed the similar level of difference in each comparison were selected and shown in FIG. 4.
For example, the genes having gene expression level of at least two times higher between the groups of non-PD vs. Parkin were 109 genes which included 20 genes showing two-fold expression in comparison of non-PD vs. Parkin and 16 genes showing two-fold expression level in all three comparisons.
Differentially-expressed genes were 413 genes where 109 genes were for non-PD vs. Parkin, 233 genes for non-PD vs. PD and 335 genes for Parkin vs. PD. Particularly, 6 genes which had been already known as PD-related gene were selected from 16 common genes in the center of non-PD vs. Parkin vs. PD, and their GENBANK accession numbers are listed in Table 4.









TABLE 4








Fold Change (log2 ratio)













GENBANK

non-PD
non-PD
Parkin



Accession
Gene name
vs.
vs.
vs.



No.
(Gene symbol)
Parkin
PD
PD
Function















NM_152753
signal peptide, CUB domain,
2.0097
−1.6452
−3.6549
protein hetero-



EGF-like 3 (SCUBE3)



homo-oligomerization


NM_000584
interleukin 8 (IL8)
−1.5026
−3.5819
−2.0792
angiogenesis/







cell motility


NM_001677
ATPase, Na+/K+ transporting, β1
1.1420
−2.1375
−3.2796
ion transport



polypeptide (ATP1B1)






NM_002546
tumor necrosis factor receptor
1.2471
−1.6420
−2.8891
apoptosis/



superfamily, member 11b(TNFRSF11B)



inflammation response


NM_004102
fatty acid binding protein 3, muscle and
−1.4108
1.7890
3.1998
phosphatidylcholine



heart (mammary-derived growth



biosynthetic process



inhibitor) (FABP3)






NM_001511
chemokine (C-X-C motif) ligand 1□□
−1.0412
−2.9751
−1.9339
chemotaxis/



(melanoma growth stimulating activity,



immune response



α) (CXCL1)









In addition, 13 genes which have been known as Parkinson's disease-related gene are selected from 56 genes in groups of non-PD vs. Parkin and Parkin vs. PD, and their GENBANK accession numbers are shown in Table 5. The genes do not include 16 genes which are common in three groups.









TABLE 5








Fold Change




(log2 ratio)












GENBANK

non-PD
Parkin



Accession
Gene name
vs.
vs.



No
(Gene symbol)
Parkin
PD
Function














NM_153262
synaptotagmin XIV
1.6903
−1.5708
membrane



(SYT14)


trafficking


NM_003667
leucine-rich
2.1567
−2.1239
G-protein



repeat-containing G


signaling



protein-coupled






receptor 5 (LGR5)





NM_003239
transforming growth
1.1171
−1.0345
cell growth/



factor, β3


aging



(TGFB3)





NM_002203
integrin α2 (ITGA2)
1.3831
−1.8448
cell adhesion


NM_004101
coagulation factor II
1.6712
−2.2182
G-protein



(thrombin) receptor-


signaling



like 2 (F2RL2)





NM_000794
dopamine receptor
1.1496
−1.1496
G-protein



D1 (DRD1)


signaling


NM_006211
proenkephalin
1.9852
−1.9852
neuropeptide



(PENK)


signaling


NM_004297
G protein α14
2.5842
−2.5842
G-protein



(GNA14)


signaling


NM_001122659
endothelin receptor
1.0755
−1.6347
G-protein



type B (EDNRB)


signaling


NM_021979
heat shock 70 kDa
1.0932
−1.4480
response to



protein 2 (HSPA2)


unfolded






protein


NM_003043
solute carrier
1.0595
−1.3053
amino acid



family 6,


metabolic



member 6


process



(SLC6A6)





NM_001628
aldo-keto reductase
1.1052
−1.3105
metabolic



family 1, member


process



B1 (aldose






reductase)






(AKR1B1)





NM_005807
proteoglycan 4
3.8210
−4.4772
cell



(PRG4)


proliferation









2.3. Clustering Analysis and Result

After finally washing and staining, the image was scanned with AFFYMETRIX GENECHIP® Human Gene 1.0 ST array using AFFYMETRIX Model 3000 G7 scanner. The image data was extracted by AFFYMETRIX Commnad Console software 1.1. The raw excel file was used for obtaining the expression extent data in the next step. The expression data was obtained by Expression Console software version 1.1. The data normalization was performed with Robust Multi-Average (RMA) algorithm in Expression Console software. The genes showing the increase of gene expression level of at least two fold between the test group and the control group were selected and used in the subsequent step.
The gene expression level of the selected genes was measured with Hierarchical clustering in MEV (MultiExperiment Viewer) software 4.0 (TM4: a free, open-source system for microarray data management and analysis. Biotechniques. 2003 February; 34(2):374-8.). To classify the genes as common gene groups having similar expression pattern, K-mean Clustering was performed (A Soukas, P Cohen, N D Socci, J M Friedman, Leptin-specific patterns of gene expression in white adipose tissue, Genes Dev, 14, 963-80 (2000)). The K-mean Clustering is a method used for classifying, based on their patterns, the common gene expression groups having the genes of similar expression pattern in hierarchical clustering analysis.
The genes which are differentially expressed were analyzed biologically with Web-based DAVID (the Database for Annotation, Visualization, and Integrated Discovery; DAVID Bioinformatics Resources 6.7, Systematic and integrative analysis of large gene lists using DAVID Bioinformatics Resources. (2009) Nat Protoc. 4(1):44-57.). The genes were classified on the basis of Gene ontology, Panther ontology database (DAVID Bioinformatics Resources 6.7).
Based on the similarity measured by the K-mean clustering analysis between non-PD, PD and Parkin groups, the expressed genes were classified. The expression pattern graphs were reorganized to seven clusters (FIG. 5b).
FIG. 5a showed a result of Hierarchical Clustering by using the signals of 413 genes which represent the difference in gene expression between the groups of PA, PD, and Parkin. The result confirmed the whole profile of clusters showing the difference in gene expression between the groups of PA, PD, and Parkin, and the expected several patterns of the clusters. FIG. 5b showed classified clusters showing similar gene expression pattern, when 7 patterns of clusters were classified according to the result of hierarchical clustering analysis. The number of pattern was determined by the smallest optimized number which was obtained after performing repetitive simulation with various pattern numbers.
Specifically, the gene names and their genbank accession number of Cluster 2, 3, 4, 5 and 6 were summarized in Tables 6a-6e.







TABLE 6a







Cluster 2: Increase













Genbank



Gene name
Gene
accession


no
(non-PD < PD = Parkin)
symbol
No





1
interleukin α8
ITGA8
NM_003638


2
cathepsin H
CTSH
NM_004390


3
chemokine (C-C motif) receptor-like 1
CCRL1
NM_178445
















TABLE 6b







Cluster 3: Increase













Genbank



Gene name
Gene
accession


no
(non-PD ≦ PD < Parkin)
symbol
No





1
transforming growth factor, β3
TGFB3
NM_003239


2
dopamine receptor D1
DRD1
NM_000794


3
G protein α14
GNA14
NM_004297


4
proenkephalin
PENK
NM_006211


5
proteoglycan 4
PRG4
NM_005807


6
leucine-rich repeat-containing
LGR5
NM_003667



G-protein coupled receptor 5




7
major histocompatibility complex,
HLA-DPA1
NM_033554



class II, DP α1
















TABLE 6c







Cluster 4: Decrease













Genbank



Gene name
Gene
accession


no
(non-PD ≧ PD > Parkin)
symbol
No





1
brain expressed, X-linked 1
BEX1
NM_018476
















TABLE 6d







Cluster 5: Decrease













Genbank



Gene name
Gene
accession


no
(non-PD > PD = Parkin)
symbol
No





1
interleukin 8
IL8
NM_000584


2
chemokine (C-X-C motif) ligand 6
CXCL6
NM_002993



(granulocyte chemotactic protein 2)
















TABLE 6e







Cluster 6: Increase













Genbank



Gene name
Gene
accession


no
(non-PD ≧ PD < Parkin)
symbol
No













1
signal peptide, CUB domain,
SCUBE3
NM_152753



EGF-like 3




2
heat shock 70 kDa protein 2
HSPA2
NM_021979


3
transforming growth factor, β3
TGFB3
NM_003239


4
dopamine receptor D1
DRD1
NM_000794


5
G protein α14
GNA14
NM_004297


6
proenkephalin
PENK
NM_006211


7
proteoglycan 4
PRG4
NM_005807


8
leucine-rich repeat-containing
LGR5
NM_003667



G-protein coupled receptor 5




9
reelin
RELN
NM_005045


10
endothelin receptor type B
EDNRB
NM_001122659


11
Integrin α2
ITGA2
NM_002203


12
solute carrier family 6, member 6
SLC6A6
NM_003043


13
coagulation factor II (thrombin)
F2RL2
NM_004101



receptor-like 2




14
cyclin-dependent kinase 6
CDK6
NM_001259


15
aldo-keto reductase family 1,
AKR1B1
NM_001628



member B1 (aldose reductase)




16
matrix metallopeptidease 8
MMP8
NM_002424


17
inhibitor of DNA binding 1,
ID1
NM_181353



dominant negative





helix-loop-helix protein




18
neurofilament, medium
NEFM
NM_005382



polypeptide




19
ATPase, Na+/K+ transporting, β1
ATP1B1
NM_001677



polypeptide




20
tumor necrosis factor receptor
TNFRSF11B
NM_002546



superfamily, member 11b




21
tumor necrosis factor receptor
TNFRSF10D
NM_003840



superfamily, member 10d, decoy





with truncated death domain









The increased pattern of gene expression was shown in Clusters 2, 3, and 6, and the decreased pattern was shown in Clusters 4 and 5. The gene data showing greatest linear-increase of gene expression (Cluster 3) and the genes showing greatest linear-decrease of gene expression (Cluster 4) could provide the numerical values of severe Parkinson's disease and a guidance for a search for the human biomarker diagnosing early-stage of Parkinson's disease.
The gene expression result is described in detail hereinafter.
Firstly, the genes which showed the linear decrease of gene expression amount in the order of PA>PD>Parkin were Cluster 4 in Table 7 and FIG. 6.










TABLE 7








Genbank



Gene name
Function
accession #








brain expressed,
multicellular organismal
NM_018476



X-linked 1
development//nervous





system development//





cell differentiation









As shown in FIG. 6, gene expression amount of gene X-linked 1 (NM—018476) showed a linear decrease in PA, PD, and Parkin. FIG. 6 is a pattern graph and Heat map showing a result of Hierarchical Clustering of the genes separated with K-mean Clustering Analysis.
The genes of Cluster 3 showed a linear increase of gene expression amount in the order of PA<PD<Parkin, as shown in Table 8 and FIG. 7.









TABLE 8







Genbank


Gene name
Function
accession #







major
antigen processing and
NM_033554


histo-
presentation of peptide or



compatibility
polysaccharide antigen via



complex, class
MHC class II//immune



II, DP alpha 1
response



MHC class I
antigen processing and
NM_000247


polypeptide-
presentation of peptide



related
antigen via MHC class I//



sequence A
response to stress//immune




response//cellular defense




response//cell recognition//




antigen processing and




presentation



pancreatic
lipid catabolic process
NM_001011709


lipase-related




protein 3




secreted
signal transduction//
NM_003014


frizzled-related
embryo implantation//Wnt



protein 4
receptor signaling




pathway//cell




differentiation



Sestrin 3
cell cycle arrest
NM_144665


EGF-like repeats
cell adhesion//multicellular
NM_005711


and discoidin
organismal development//



I-like domains 3
angiogenesis



aldo-keto
prostaglandin metabolic
NM_003739


reductase family
process



1, member C3




(3-alpha




hydroxysteroid




dehydrogenase,




type II)









FIG. 7 is a result of clustering of the genes which showed a linear increase of gene expression in non-PD, PD, and Parkin.
2.4. Reprogramming of Functional Group of Human Biomarker Candidate Among Non-PD Vs. PD and Non-PD Vs. Parkin
The functional groups among non-PD vs. PD and non-PD vs. Parkin were reprogrammed, and the human biomarker candidates were classified by using the genes obtained from Gene Ontology and Panther database system (DAVID Bioinformatics Resources 6.7) (FIGS. 8a to 8d). FIGS. 8a to 8d represented the number of genes which were reclassified as biologically-functional groups from the genes having the gene expression level of at least two-fold between the three groups of non-PD, PD, Parkin.
The biological categories included transcription factor, nucleic acid binding, receptor, kinase, oxido-reduction protein, signal molecule, cell adhesion molecule and the like.
The genetic functional groups which showed up-regulation (FIG. 8a) or down-regulation (FIG. 8b) in Idiopathic PD patient were compared with those of non-PD patient (control group). The genetic functional groups which showed up-regulation (FIG. 8c) or down-regulation (FIG. 8d) in Parkin (parkin deficiency) patient were compared with those of non-PD patient (control group). These graphs represented human biomarker candidate which were re-classified according to the biological functions and notable up-regulation or down-regulation in idiopathic PD patient and parkin-deficiency PD patient.
2.5. Genes Regulated Differentially in Non-PD, PD and Parkin Patients Due to the Oxidative Stress
It has not been known which genes show selective sensitivity to the oxidative stress and how those genes affect the cell. PD-related genes which are differentially regulated by the oxidative stress are analyzed in non-PD, PD and Parkin groups. The genes regulated differentially by the oxidative stress were classified again as a functional group from the genes showing the gene expression level of at least two-fold between the groups of non-PD, PD, and Parkin, and then the PD-related genes were selected.
These groups included oxidoreductase, endoplasmic reticulum/ubiquitin-like, exocytosis/membrane trafficking, apoptosis/cell survival, structure/transport, translation, nuclear/transcriptional, and cell cycle. The PD-related genes in K-mean clustering 2 and 6 were classified again to change the cluster number. The genes which were differentially expressed between non-PD, PD and Parkin because of the oxidative stress are summarized in Table 9.









TABLE 9







K-mean



Putative Function
clustering








Group I: oxidoreductase




aldo-keto reductase family 1, member
6



B1 (aldose reductase) (AKR1B1)




Group II: endoplasmic




reticulum/ubiquitin-like




dopamine receptor D1 (DRD1)
6



Group III: exocytosis/membrane trafficking




major histocompatibility complex,
3



class II, DP α1 (HLA-DPA1)




synaptotagmin XIV (SYT14)
6



Group IV: apoptosis/cell survival




actin, α, cardiac muscle 1 (ACTC1)
2



clusterin (CLU)
2



transforming growth factor, β3 (TGFB3)
6



Group V: structure/transport




major histocompatibility complex,
3



class II, DP α1 (HLA-DPA1)




transforming growth factor, β3 (TGFB3)
6



solute carrier family 6, member 6 (SLC6A6)
6



aldo-keto reductase family 1, member B1
6



(aldose reductase) (AKR1B1)




signal peptide, CUB domain, EGF-like 3
6



(SCUBE3)




Group VI: translation




angiotensin II receptor, type 1 (AGTR1)
2



reelin (RELN)
6



G protein α14 (GNA14)
6



solute carrier family 6, member 6 (SLC6A6)
6



Group VII: nuclear/transcriptional




integrin α2 (ITGA2)
6



transforming growth factor, β3 (TGFB3)
6



dopamine receptor D1 (DRD1)
6



Group VIII: cell cycle




heat shock 70 kDa protein 2 (HSPA2)
6









Surprisingly, the selective gene expression of the groups in Clusters 2, 3, and 6 increased linearly between non-PD, PD and Parkin patients, and the groups belonged to AKR1B1 (oxidoreductase), DRD1 (endoplasmic reticulum/ubiquitin-like), HLA-DPA1 and SYT14 (exocytosis/membrane trafficking), ACTC1, CLU and TGFB3 (apoptosis/cell survival), HLA-DPA1, TGFB3, SLC6A6, AKR1B1 and SCUBE (structure/transport), AGTR1, RELN, GNA14 and SLC6A6 (translation), ITGA2, TGFB3 and DRD1 (nuclear/transcriptional), and HSPA2 (cell cycle). The GENBANK accession numbers of the genes are summarized in the following table.













Gene name
Gene accession number








AKR1B1
NM_001628



DRD1
NM_000794



HLA-DPA1
NM_033554



SYT14
NM_153262



ACTC1
NM_005159



CLU
NM_001831



TGFB3
NM_003239



SLC6A6
NM_003043



SCUBE3
NM_152753



AGTR1
NM_000685



RELN
NM_005045



GNA14
NM_004297



ITGA2
NM_002203



HSPA2
NM_021979









The obtained data can assist the understanding of mitochondrial dysfunction and oxidative stress in Idiopathic and Parkin-derived Parkinson's disease, and provide a useful guidance for investigation of additional functional properties.
2.6. Immortalization of Mesenchymal Stromal Cells Derived from Adipose Tissue of Parkinson's Disease Patient with pGRN145 Including hTERT
The cells of non-PD, PD, Parkin in Example 1.1 were spread again on the 24-well plate to reach 90 percent of confluence without adding antibiotics on one day before transfecting. 50 μL of serum-free OPTI-MEM I Medium (Gibco BRL, Gaithersburg, Md.) including 1 μg of pGRN145 DNA (Geron Corporation, Menlo Park, Calif., USA), and 50 μL of OPTI-MEM I Medium including 2 mL of LIPOFECTAMINE LTX Reagent (Gibco) were mixed and added to each well, and replaced with new media after culturing at 37° C. for 24 hr. After 48 hours, the transfected cells were cultured in media including Hygromycin-B (30 μg/mL) for 2 to 3 weeks, and the final concentration was reduced to be 10 μg/mL. The clones derived from one cell were selected.
The cell shapes of non-PD, PD, and Parkin cells belonging to the selected clones were compared before immortalization, after immortalization, 6-month culture and one-year culture with human telomerase reverse transcriptase (hTERT).
The objected immortalized cells were deposited at Korean cell line bank located at 28 Yungdon-dong, Chongrno-gu, Seoul 110-799, Republic of Korea on Nov. 17, 2010, and then assigned with the accession numbers of KCLRF-BP-00239 (PA1), KCLRF-BP-00240 (PA2), KCLRF-BP-00241 (PD1), KCLRF-BP-00242 (PD2), KCLRF-BP-00243 (Pakin1), and KCLRF-BP-00244 (Pakin2).
The result is described in FIG. 9. In FIG. 9, non-PD (PA) represents the shape of cell having Accession No. KCLRF-BP-00239 (PA1) before and after immortalization, PD is for the shape of cell having Accession No. KCLRF-BP-00241 (PD1) before and after immortalization, and Parkin is for the shape of cell having Accession No. KCLRF-BP-00243 (Pakin1) before and after immortalization.
The chromosomal structure of the cells obtained shortly after the immortalization and after culturing the immortalized non-PD, PD, and Parkin for a year were analyzed and shown in FIG. 10. The chromosomal structure of the cells before immortalization was normal, and thus was not analyzed. From top to bottom in FIG. 10, non-PD (PA) represents the state of immortalized cell having Accession Nos. KCLRF-BP-00239 (PA1) and KCLRF-BP-00240 (PA2), PD represents the state of immortalized cell having Accession Nos. KCLRF-BP-00241 (PD1) and KCLRF-BP-00242 (PD2), and Parkin represent the state of immortalized cell having Accession Nos. KCLRF-BP-00243 (Pakin1) and KCLRF-BP-00244 (Pakin2).
Specifically, the cell division at metaphase of mitosis was restrained with colcemid (Gibco) Stoc solution. That is, the cells were collected from the supernatant obtained by centrifuging at 1500 rpm, shocked with 0.075M KCl hypotonic, and fixed with the addition of Canoy's fixative including methanol and acetic acid at a mixing ratio of 3:1, and Giemsa staining GTG banding). The prepared cell slide was analyzed with Karyotype Analysis program: ChIPS-Karyo (Chromosome Image Processing System) (GenDix, Inc. Seoul, Korea), and the analyzed result is shown in FIG. 10.
As shown in FIG. 10, the immortalized cell showed abnormal karyotype compared with the non-immortalized cell.
2.7. Separation of Mitochondria from the Cultured Cell for Mitochondria Complex I, II, IV and Citrate Synthase Assays
The non-PD, PD, and Parkin cells immortalized with hTERT were washed with PBS and suspended in 10 mM Tris, pH 7.6 including protease inhibitor cocktail. The cells were blocked with 1-mL syringe, added with 1.5M sucrose and centrifuged at 600×g, 2° C. for 10 minutes. Then, the supernatant were centrifuged again at 14,000×g, 2° C. for 10 minutes and the obtained pellet were washed with protease inhibitor cocktail in 10 mM Tris (pH 7.6). The mitochondria pellets were re-suspended in 10 mM Tris (pH 7.6) including protease inhibitor cocktail and subsequently preserved in ice before use.
Complex I Assay:
The activity of complex I was analyzed with spectrometer at 600 nm by using 240 μL reagent including 25 mM potassium phosphate, 3.5 g/L BSA, 60 μM DCIP, 70 μM decylubiquinone, 1.0 μM antimycine-A, and 3.2 mM NADH, pH 7.8.
Namely, the obtained mitochondria sample (1 μg/10 μL) was added to a buffer solution without NADH, incubated at 37° C. for 3 minutes, and then added with 5 μL of 160 mM NADH. The absorbance was measured at 37° C. for 5 minutes at 30 second-intervals, and after 5 minutes, and 2.5 μL rotenone (100 μM of rotenone dissolved in 1 mM in dimethylsulfoxide and 10 mM Tris, pH 7.6) was added thereto. Then, the absorbance was measured at 37° C. for 5 minutes at 30 second-intervals. The results are shown in FIG. 11.
Complex II Assay:
The activity of complex II was analyzed with spectrometer at 600 nm with 240 μL reagent including 80 mM potassium phosphate, 1 g/L BSA, 2 mM EDTA, 0.2 mM ATP, 10 mM succinate, 0.3 mM potassium cyanide, 60 μM DCIP, 50 μM decylubiquinone, antimycine-A, and 3 μM rotenone, pH 7.8.
Specifically, the obtained mitochondria sample (1 μg/10 μL) was added to a buffer solution without succinate and potassium cyanide, incubated at 37° C. for 10 minutes, and then 20 μL of 1.5M succinate and 0.75 μL of 0.1M KCN were added thereto. The absorbance was measured at 37° C. for 5 minutes at 30 second-intervals, and BLANK was detected in the presence of 5 mM malonate. The result is shown in FIG. 11.
Complex IV Assay:
The activity of complex IV was analyzed with spectrometer at 550 nm with 240 μL reagent including 30 mM potassium phosphate, 2.5 mM dodecylmaltoside, and 34 μM ferrocytochrome c, pH 7.4
Specifically, the obtained mitochondria sample (1 μg/10 μL) was added to a buffer solution and shortly after, the absorbance was measured at 30° C. for 5 minutes at 30 second-intervals, and BLANK was detected in the presence of 1 mM KCN. The result is shown in FIG. 11.
Citrate Synthase Assay:
The activity of citrate synthase was analyzed with spectrometer at 412 nm with 2404 reagent (pH 7.5) including 50 mM Tris-HCl, 0.2 mM 5,5′-dithiobis-(2-nitrobenzoic acid), 0.1 mM acetyl-CoA and 0.5 mM oxaloacetate.
Specifically, the obtained mitochondria sample (1 μg/10 μL) was added to a buffer solution without oxaloacetate, and incubated at 30° C. for 5 minutes. After adding by 2.5 μL of 50 mM oxaloacetate, the absorbance was measured at 37° C. for 5 minutes at 30 second-intervals. The result is shown in FIG. 11. This assay began with the addition of oxaloacetate, and for a control group, water was added in the equal amount.
As represented in FIG. 11, the result of biochemical analysis for mitochondrial respiration chain of immortalized cell confirmed that the activities of PD and Parkin decreased compared with Non-PD activity.
2.8. Electron Microscopy Analysis
The non-PD, PD, and Parkin cells immortalized with hTERT were washed with PBS and fixed with 0.1% glutaraldehyde and 4% paraformaldehyde in PBS at 4° C. for 2 hours. The cells were collected and centrifuged at 2000×g, at 4° C. and for 3 minutes to obtain pellets. The prepared pellets were re-suspended in warm 1% agar and centrifuged at 2000×g at 4° C. for 3 minutes to obtain the pellets again. Then, the pellets were washed with PBS three times, and the cell pellets embedded with agar were fixed again with 1% osmium tetroxide for 2 hours and washed with PBS three times. The cell pellets embedded with agar was dehydrated in ethanol and fixed again with Epon 812. The ultrathin (70 nm) sections were collected on Formvar/carbon-coated nickel grids, stained with 2.5% uranyl acetate for 7 minutes and with lead citrate for 2.5 minutes, and then were observed with JEOL JEM-1011 electron microscope. The results are shown in FIG. 12. The comparison of mitochondrial shape of primary-cultured non-PD, PD and Parkin cell and immortalized non-PD, PD and Parkin cell showed that the mitochondria shape of non-PD (non-Parkinson's disease) patient was normal, but those of PD (Idiopathic Parkinson's disease) patient and Parkin (parkin-deficient Parkinson's disease) patient were damaged gradually. This suggested the mitochondrial damage is an important cause of Parkinson's disease.
2.9. Western Blot Analysis
The cells (The primary-cultured mesenchymal stromal cells derived from non-PD, PD, and Parkin patients were used in FIG. 13a, and immortalized mesenchymal stromal cells derived from non-PD, PD, and Parkin patients were used in FIGS. 13b, 13c, 14a, 15a, and 16a) were washed with cold PBS and divided into lysis buffer (cell signaling) and PMSF. The divided products were centrifuged at 15,000×g at 4° C. for 20 minutes. The products were analyzed quantitatively with Bradford reagent (Bio-Rad, Hercules, Calif.). The protein which was used in the same amount as other primary-cultured cells, and immortalized cells were loaded on SDS-PAGE, transferred to PVDF membrane (Millipore), and blocked with 5% non-fat dry milk in TBST. The proteins on the membrane were detected by chemical luminescence with X-ray film using ECL-Plus substrate (GE Healthcare, Buckinghamshire, USA). Antibodies of Hsp25, Hsp60 and Hsp90 were obtained from Santa Cruz Biotechnology (Santa Cruz, Calif., USA) and antibodies of DJ-1, P-mTOR, mTOR, P-S6K, S6K, LC3-I, and LC3-II were obtained from Chemicon (Temecula, Calif., USA). Prohibitin and β-actin (Santa Cruz) were used as internal control. The western blot analysis was performed with National Institutes of Health image processing and analyzing program (ImageJ, v1.38).
The obtained results are shown in FIGS. 13a-13c, 14a-14c, 15a-15b and 16a-16b. FIG. 13a represents the change in the gene expression of mitochondrial markers, DJ-1, Hsp60, and Hsp25 in the primary-cultured mesenchymal stromal cells derived from non-PD, PD, and Parkin patients. FIG. 13b represents the change in the gene expression of mitochondrial markers, Hsp60 in immortalized mesenchymal stromal cells derived from non-PD, PD, and Parkin patients. FIG. 13c represents the change in the gene expression of mitochondrial markers, Hsp90 in immortalized mesenchymal stromal cells derived from non-PD, PD, and Parkin patients. FIG. 14a represents the change in the gene expression of autophagy marker, mTOR, and S6K in immortalized mesenchymal stromal cells derived from non-PD, PD, and Parkin patients. FIG. 14b and FIG. 14c are the graphs showing the quantitative analysis of FIG. 14a. FIG. 15a represents the change in the gene expression of autophagy marker, mTOR, and S6K in immortalized mesenchymal stromal cells derived from non-PD, PD, and Parkin patients. FIG. 15b is the graph showing the quantitative analysis of FIG. 15a. FIG. 16a represents the change in the gene expression of autophagy marker, LC3 (LC3-I, LC3-II) in immortalized mesenchymal stromal cells derived from non-PD, PD, and Parkin patients. FIG. 16b is the graph showing the quantitative analysis of FIG. 16a.

## Claims

1. An immortalized mesenchymal stromal cell, which is prepared by immortalizing a mesenchymal stromal cell separated from human adipose tissue of a patient without Parkinson's disease using human telomerase reverse transcriptase, and has an abnormal karyotype, wherein the immortalized mesenchymal stromal cell is a cell deposited with the Korean Cell Line Research Foundation under Accession No. KCLRF-BP-00239 or KCLRF-BP-00240.

2. An immortalized mesenchymal stromal cell, which is prepared by immortalizing a mesenchymal stromal cell separated from human adipose tissue of patient with Idiopathic Parkinson's disease using human telomerase reverse transcriptase, and has an abnormal karyotype, wherein the immortalized mesenchymal stromal cell is a cell deposited with the Korean Cell Line Research Foundation under Accession No. KCLRF-BP-00241 or KCLRF-BP-00242.

3. The immortalized mesenchymal stromal cell of claim 2, which has decreased mitochondrial activity compared with that of the immortalized mesenchymal stromal cell deposited with the Korean Cell Line Research Foundation under Accession No. KCLRF-BP-00239 or KCLRF-BP-00240.

4. An immortalized mesenchymal stromal cell, which is prepared by immortalizing a mesenchymal stromal cell separated from human adipose tissue of patient with Parkin-deficient Parkinson's disease using human telomerase reverse transcriptase, and has an abnormal karyotype, wherein the immortalized mesenchymal stromal cell is a cell deposited with the Korean Cell Line Research Foundation under Accession No. KCLRF-BP-00243 or KCLRF-BP-00244.

5. The immortalized mesenchymal stromal cell of claim 4, which has decreased mitochondrial activity compared with that of the immortalized mesenchymal stromal cell deposited with the Korean Cell Line Research Foundation under Accession No. KCLRF-BP-00239 or KCLRF-BP-00240.