ENHANCED POSTNATAL ADHERENT CELL, AND USE FOR SAME

Abstract

The present invention relates to an enhanced postnatal adherent cell (ePAC), a method for producing same, and a composition and a cell therapeutic agent having same as an active ingredient.

Claims

1. An enhanced postnatal adherent cell having one or more characteristics selected from the group consisting of (a) to (h): (a) maintaining a morphology of adherent fibroblasts during subculture; (b) having ability to differentiate into adipocytes, osteocytes, or chondrocytes; (c) secreting one or more proteins selected from the group consisting of VEGF, TGF-1, IL-6, progranulin, follistatin, angiostatin, MMP2, MMP10, TRAIL R2, and MMP3; (d) having surface antigen characteristics of HLA-G, CD34, MIC A/B+, CD200+, CD61+, CD130+, CD321+, SSEA4+, and CD338+; (e) having high expression levels of one or more genes selected from the group consisting of COL3A1, IGFBP5, PRNP and MT1A, as compared with bone marrow-derived stem cells; (f) having low expression levels of one or more genes selected from the group consisting of COL1A1, COL1A2, TPM2, TAGLN, CYP1B1, CXCL12, and PENK, as compared with bone marrow-derived stem cells; (g) having high expression levels of one or more proteins selected from the group consisting of LIN28B, FERMT3, RAB27B and PEG10, as compared with bone marrow-derived stem cells; and (h) having low expression levels of HYOU1 or GLIPR1 protein, as compared with bone marrow-derived stem cells.

2. The enhanced postnatal adherent cell of claim 1, having the characteristics of (e), (f), or both, wherein the characteristics of (e) and (f) show that the expression level difference from those of bone marrow-derived stem cells is twice or more, as measured by microarray analysis.

3. The enhanced postnatal adherent cell of claim 1, wherein the cell is derived from amnion.

4. The enhanced postnatal adherent cell of claim 1, wherein when the cell is cultured under a hypoxia condition or under a normoxia condition it has the following characteristics: the cell cultured under the hypoxia condition has increased expression levels of one or more genes selected from the group consisting of PGK1, BNIP3, TPI1, ERRFI1, LOC644774, SLC2A3, PLIN2, and TUBB2B, as compared with the cell cultured under the normoxia condition; the cell cultured under the hypoxia condition has decreased expression levels of one or more genes selected from the group consisting of SERPINE1, TAGLN, TGM2, IL-8, and ALDH1A3, as compared with the cell cultured under the normoxia condition; the cell cultured under the hypoxia condition has increased expression levels of one or more proteins selected from the group consisting of PODXL, HIST1H1B, and PLEK2, as compared with the cell cultured under the normoxia condition; or the cell cultured under the hypoxia condition has decreased expression level of DPP4 protein, as compared with the cell cultured under the normoxia condition.

5. A population of enhanced postnatal adherent cells, wherein the postnatal adherent cells have the following characteristics: (a) maintaining a morphology of adherent fibroblasts during subculture; (b) having ability to differentiate into adipocytes, osteocytes, or chondrocytes; (c) secreting one or more proteins selected from the group consisting of VEGF, TGF-1, IL-6, progranulin, follistatin, angiostatin, MMP2, MMP10, TRAIL R2, and MMP3; (d) having surface antigen characteristics of HLA-G, CD34, MIC A/B+, CD200+, CD61+, CD130+, CD321+, SSEA4+, and CD338+; (e) having a high expression level of one or more genes selected from the group consisting of COL3A1, IGFBP5, PRNP and MT1A, as compared with bone marrow-derived stem cells; (f) having a low expression level of one or more genes selected from the group consisting of COL1A1, COL1A2, TPM2, TAGLN, CYP1B1, CXCL12, and PENK, as compared with bone marrow-derived stem cells; (g) having a high expression level of one or more proteins selected from the group consisting of LIN28B, FERMT3, RAB27B and PEG10, as compared with bone marrow-derived stem cells; and (h) having a low expression level of HYOU1 or GLIPR1 protein, as compared with bone marrow-derived stem cells.

6. The population of enhanced postnatal adherent cells of claim 5, wherein the cells in the population are derived from amnion.

7. The population of enhanced postnatal adherent cells of claim 5, wherein the cells in the population secrete progranulin.

8. The population of enhanced postnatal adherent cells of claim 5, wherein 70% or more of the cells in the population express CD200.

9. A method of preparing an enhanced postnatal adherent cell, comprising reacting an amnion tissue separated from a chorionic plate membrane of placenta with an enzyme mixture, wherein the cell has the following characteristics: (a) maintaining a morphology of adherent fibroblasts during subculture; (b) having ability to differentiate into adipocytes, osteocytes, or chondrocytes; (c) secreting one or more proteins selected from the group consisting of VEGF, TGF-1, IL-6, progranulin, follistatin, angiostatin, MMP2, MMP10, TRAIL R2, and MMP3; (d) having surface antigen characteristics of HLA-G, CD34, MIC A/B+, CD200+, CD61+, CD130+, CD321+, SSEA4+, and CD338+; (e) having high expression levels of one or more genes selected from the group consisting of COL3A1, IGFBP5, PRNP and MT1A, as compared with bone marrow-derived stem cells; (f) having low expression levels of one or more genes selected from the group consisting of COL1A1, COL1A2, TPM2, TAGLN, CYP1B1, CXCL12, and PENK, as compared with bone marrow-derived stem cells; (g) having high expression levels of one or more proteins selected from the group consisting of LIN28B, FERMT3, RAB27B and PEG10, as compared with bone marrow-derived stem cells; and (h) having low expression levels of HYOU1 or GLIPR1 protein, as compared with bone marrow-derived stem cells.

10. The method of claim 9, wherein the enzyme mixture comprises one or more selected from the group consisting of trypsin, dispase, collagenase, and DNAase.

11. The method of claim 9, further comprising isolating the enhanced postnatal adherent cell by reacting an animal component-free recombinant enzyme with the cell recovered after reaction of the amnion tissue and the enzyme mixture.

12. An enhanced postnatal adherent cell prepared by the method of claim 9.

13. A composition comprising the enhanced postnatal adherent cell of claim 1.

14. A pharmaceutical composition comprising the enhanced postnatal adherent cell of claim 1 as an active ingredient suitable for treating or preventing a neurodegenerative disease.

15. The pharmaceutical composition of claim 14, wherein the neurodegenerative disease is one or more selected from the group consisting of Alzheimer's disease, concussion, stroke, Parkinson's disease, Pick's disease, Huntington's disease, progressive supranuclear palsy, multiple sclerosis, amyotrophic lateral sclerosis (ALS), dementia, and traumatic brain injury.

16. An agent comprising the enhanced postnatal adherent cell of claim 1.

17. A cell therapeutic agent comprising the enhanced postnatal adherent cell of claim 1 as an active ingredient.

18. (canceled)

19. A method of treating or preventing a neurodegenerative disease, the method comprising administering the enhanced postnatal adherent cell of claim 1, cell populations thereof, or a culture thereof as an active ingredient to a subject in need thereof.

Description

DESCRIPTION OF THE DRAWINGS

[0107] FIG. 1 shows results of analyzing Oct4a and Nanog expression patterns of embryonic stem cells (ES) and enhanced postnatal adherent cells (ePACs) by immunocytochemistry;

[0108] FIG. 2 shows a morphology of ePACs observed by using an inverted microscope (Eclipse TS100 (Nikon) at 100 magnification;

[0109] FIG. 3 shows immune-modulating ability of ePACs, in which the graph represents inhibition rates of T cell proliferation when monocytes isolated from the peripheral blood were directly or indirectly co-cultured with ePACs;

[0110] FIG. 4 shows immune-modulating ability of ePACs according to subculturing, in which the graph represents inhibition rates of T cell proliferation when monocytes isolated from the peripheral blood were directly or indirectly co-cultured with ePACs at passage 1 (P1), passage 3 (P3), or passage 6 (P6);

[0111] FIG. 5 shows multipotency of ePACs of the present disclosure, in which the ePACs of the present disclosure differentiated to adipocytes, osteocytes, or chondrocytes under both conditions of hypoxia and normoxia;

[0112] FIG. 6 shows migration ability of ePACs cultured under hypoxia and normoxia conditions, n=3, * P<0.06;

[0113] FIG. 7 shows colony forming ability of ePACs cultured under hypoxia and normoxia conditions, n=3, * P<0.06;

[0114] FIG. 8 shows characteristics of ePACs of the present disclosure according to culture passages, in which A represents a cumulative population doubling level (CPDL) according to each cell passage of ePACs, B represents a population doubling level (PDL) according to each cell passage of ePACs, and C represents a cell size of ePACs according to each cell passage;

[0115] FIG. 9 shows results of ELISA for analyzing secretion of VEGF, TGF-1, progranulin, and IL-6 from ePACs at each passage, after examining secretory proteins of ePACs by Ab arrays;

[0116] FIG. 10 shows results of ELISA for analyzing secretion of HGF from ePACs at each passage;

[0117] FIG. 11 are graphs showing results of flow cytometry for analyzing immunological characteristics of ePACs for HLA-ABC, HLA-DR, and HLA-G and other surface proteins;

[0118] FIG. 12 shows results of comparing and analyzing protein expressions of ePACs according to a specific embodiment; and

[0119] FIG. 13 shows results of analyzing T cell proliferation-inhibitory effect (A) and neuronal cell-proliferating effect (B) of ePACs according to a specific embodiment.

MODE OF THE INVENTION

[0120] Hereinafter, the present invention will be described in more detail. However, these Examples are for illustrative purposes only, and the scope of the present invention is not intended to be limited by these Examples.

Example 1. Preparation of Enhanced Postnatal Adherent Cells

[0121] 1.1. Isolation of Adherent Cells from Amnion Tissue of Placenta

[0122] A placenta of a healthy woman who had normally delivered was used, after an informed consent form was signed by the woman who had been given information about the research. A chorionic plate membrane was pulled and peeled off from a placental tissue collected during normal placenta delivery. The removed chorionic plate membrane was washed with Ca/Mg free DPBS containing gentamicin twice or five times to remove blood, and then a chorionic plate was scraped and removed by using a slide glass. The remaining amnion was divided into pieces as small as possible using surgical scissors such that the pieces was in a size of about 1 mm to about 5 mm, and 20 ml of an enzyme reaction solution (enzyme mixture) was added to the small tissue pieces and allowed to react in a shaking incubator at 37 C. and 200 rpm for 15 minutes. Components and concentrations of the used enzyme reaction solution are given in the following Table 1. To inactivate the enzyme reaction solution, 2 ml of FBS was added at a ratio of 1:10, and the reaction solution was centrifuged at 1,500 rpm for 3 minutes, and then a supernatant was transferred to a new tube. This procedure was repeated twice with respect to the remaining tissues. From the dissolved tissue, amniotic cells were isolated using a 100-m cell strainer.

TABLE-US-00001 TABLE 1 Component Concentration Available source HBSS (Hank's Balanced Invitrogen Salt Solution) Trypsin 1.8 mg/ml Sigma Dispase 1 U/ml Invitrogen Collagenase I 1.2 mg/ml Invitrogen DNase I 25 g/ml Rhoche

[0123] 1.2. Culture of Isolated Adherent Cells

[0124] (1) Culture Under Hypoxia Condition

[0125] The amniotic cells isolated in Example 1.1 were centrifuged and a supernatant was removed therefrom. Cell pellets were suspended in a PS-CM medium, and seeded in a T-flask, and then cultured at 37 C. under a hypoxia condition (CO.sub.2 5%, O.sub.2 3%). The cells were cultured until cell colonies were formed to occupy 50%80% of the bottom area of T-flask. Every 3 days to 4 days, PS-CM medium was replaced to remove cells which did not adhere to the bottom of flask. Components of the PS-CM medium are given in the following Table 2. Only cells isolated by treatment of TrypLE, which is an animal component-free (ACF) recombinant enzyme (Invitrogen), in a 37 C. incubator for a short time (3 minutes) at a first passage, were used to increase purity of amnion-derived adherent cells.

TABLE-US-00002 TABLE 2 Components of PC-CM medium Component Concentration Available source MEM alpha GlutaMAX Invitrogen Fetal Bovine Serum (FBS) 10% Invitrogen Fibroblast growth factor 4 25 ng/ml Peprotech (FGF4) Heparin 1 g/ml Sigma Gentamicin 50 g/ml Invitrogen

[0126] (2) Culture Under Normoxia Condition

[0127] The amniotic cells isolated in Example 1.1 were cultured in the same manner as in the culture under hypoxia condition in Example 1.2.(1) except that an oxygen concentration was changed during culture. The oxygen concentration during culture was 21%, and the same PC-CM medium as in Example 1.2.(1) was used.

Example 2. Characterization of Prepared Enhanced Postnatal Adherent Cells

[0128] 2.1. Examination of Cell Morphology

[0129] Morphology and culture characteristics of the enhanced postnatal adherent cells cultured in Example 1 were observed by using an inverted microscope (Eclipse TS100 (Nikon)) at 100 magnification.

[0130] FIG. 2 is a microscopic image of the cells, and the enhanced postnatal adherent cells of the present disclosure had a morphology specific to fibroblasts with irregular protrusions and proliferated while adhering to a plastic ware. The enhanced postnatal adherent cells of the present disclosure were smaller than known placenta-derived mesenchymal stem cells, and had a homogenous cell morphology.

[0131] 2.2. Analysis of Surface Antigen Expression

[0132] (1) Flow Cytometric Analysis

[0133] To analyze characteristics of the prepared amnion-derived adherent cells, each 110.sup.6 of the cells cultured under hypoxia and normoxia conditions of Example 1 were collected in a 1.5 ml tube, and reacted with fluorescent-labeled CD1a, CD3, CD8a, CD11c, CD14, CD16, CD19, CD31, CD34, CD40, CD44, CD45, CD56, CD61, CD73, CD80, CD86, CD90, CD105, CD106, CD130, CD166, CD200, CD321, CD338, PDGFR, PDGFR, TRA-1-60, HLA-DR, HLA-G, HLA-ABC, MIC A/B and SSEA4 antibodies, respectively. Cell-specific surface antigen expressions were analyzed by using a flow cytometer (Facs Caliber, BD science), and the results are shown in FIG. 11.

[0134] As a result, all of the cells cultured under hypoxia and normoxia conditions showed negative characteristics for CD1a, CD3, CD8a, CD11c, CD16, CD19, CD31, CD34, CD40, CD45, CD56, CD80, CD86, PDGF, TGA-1-60, HLA-DR, and HLA-G, and positive characteristics for CD44, CD61, CD73, CD90, CD105, CD130, CD166, CD200, CD321, CD338, SSEA4, PDGFR, and HLA-ABC. Meanwhile, 10% or more of the cells showed CD106 expression and 70% or more of the cells showed CD200 expression (see FIG. 11).

[0135] Oct4 and Nanog expression patterns in the cells cultured in Example 1 were examined by immunocytochemistry (ICC). Embryonic stem cells (ES) were used as a positive control group and Oct4 and Nanog expression patterns at P1 and P6 of the cells were analyzed.

[0136] Specifically, the collected cells were washed with DPBS three times, and then fixed in PBS containing 4% paraformaldehyde for 10 minutes. The cells were washed with DPBS three times, and allowed to react with 0.2% Triton X-100 solution at room temperature for 10 minutes, and washed with DPBS three times. Blocking was performed by using 10% normal goat serum at room temperature for 30 minutes. Stem cell markers, Oct4 and Nanog, which are primary antibodies, were added and allowed to react overnight at 4 C. in the dark. Thereafter, the cells were washed with DPBS three times, and secondary goat antibodies were added thereto, and allowed to react in the dark for 1 hour. Lastly, the cells were washed with DPBS three times, and observed under a fluorescence microscope (FIG. 1).

[0137] As a result, all the enhanced postnatal adherent cells of the present disclosure cultured under hypoxia and normoxia conditions showed negative characteristics for Oct4 and Nanog which function to maintain progenitor cell characteristics of embryonic stem cells (FIG. 1), indicating loss of possibility of teratoma formation. Accordingly, it is expected that the cells of the present disclosure have high stability when they are applied to cell therapeutic agents.

Example 3. Profiling and Quantification Analysis of Secretory Proteins of Enhanced Postnatal Adherent Cells

[0138] 3.1. Profiling of Secretory Proteins

[0139] Profiling of secretory proteins was performed in order to analyze secretory proteins of the cells prepared in Example 1. In detail, the adherent cells cultured under normoxia and hypoxia conditions until confluency reached 90% were allowed to secrete secretory proteins in serum-free MEM alpha GlutaMAX (Invitrogen), and secretory proteins were concentrated at a concentration of 1 mg/ml. The concentrate was reacted with a membrane (human antibody array (Raybio)) capable of analyzing 504 kinds of secretory proteins, and fluorescence was developed to examine proteins secreted from the adherent cells. As shown in the following Table 3, a total of 54 kinds of proteins were found to be secreted.

TABLE-US-00003 TABLE 3 Cytokine 1 Thrombospondin (TSP) 2 EDA-A2 3 IGFBP-rp1/IGFBP-7 4 Thrombospondin-1 5 MMP-1 6 HGF 7 IL-8 8 sgp130 9 WIF-1 10 IL-6 11 TIMP-2 12 GRO 13 Latent TGF-beta bp1 14 GDF-15 15 sFRP-4 16 IL-19 17 Kremen-2 18 TGF-beta RIII 19 M-CSF 20 MSP alpha Chain 21 MIP 2 22 TNF RI/TNFRSF1A 23 MCP-3 24 Galectin-3 25 MCP-1 26 sFRP-1 27 TGF-beta RI/ALK-5 28 IL-15 R alpha 29 ENA-78 30 IL-7 31 SPARC 32 VEGF 33 Inhibin B 34 IGFBP-3 35 Nidogen-1 36 EMAP-II 37 Progranulin 38 MIF 39 IL-3 40 IGFBP-6 41 TIMP-1 42 IGF-II R 43 Activin C 44 Smad 4 45 Decorin 46 Dkk-1 47 MIP-1a 48 FGF-7/KGF 49 Follistatin 50 Angiostatin 51 MMP2 52 MMP10 53 TRAIL R2 54 MMP3

[0140] 3.2. Quantification Analysis of Secretory Proteins

[0141] Among the secretory proteins, VEGF, TGF-1, progranulin, HGF, and IL-6 which are known as useful proteins for neurological diseases, immune diseases, or vascular diseases were found to be secreted at a large amount, and quantification analysis thereof was performed according to a donor and a passage.

[0142] The amounts of the five secretory proteins were analyzed by an enzyme-linked immunosorbent assay (ELISA). An equal number of the cells at each passage (see FIGS. 9 and 10) was seeded in a 6-well plate and cultured for 1 day. Then, the medium was replaced by serum-free MEM alpha GlutaMAX (Invitrogen) and cultured for 1 days. This culture was used as a sample. As in the following Table 4, respective ELISA kits were used, and among them, TGF-1 includes a pretreatment process of the sample. All were measured at 450 nm by using a microplate reader Epoch (BioTek Inc.), and analyzed by using a Gen5 (2.00) software, and the results are shown in FIGS. 9 and 10.

TABLE-US-00004 TABLE 4 ELISA Kit Product No. Available source Human VEGF DVE00 R&D system Human TGF-1 DB100B R&D system Human progranulin DPGRN0 R&D system Human HGF SEA047Hu Uscn life Science Inc. Human IL-6 D6050 R&D system

[0143] As a result, VEGF secretion was increased with increasing passage, and TGF-B1 was constantly secreted regardless of the donor or passage. Further, progranulin which is a protein useful for improvement of neurological diseases showed no great difference in the secretion according to the donor or passage. IL-6 expression levels were increased with increasing passage, and showed a difference according to the donor. HGF was secreted at a small amount and there was no difference according to the donor or passage.

Example 4. Profiling and Quantification Analysis of Secretory Proteins of Enhanced Postnatal Adherent Cells

[0144] 4.1. Analysis of Multipotency

[0145] Differentiation ability of the adherent stem cells isolated in Example 1 into adipocytes, osteocytes, or chondrocytes was tested under hypoxia and normoxia conditions.

[0146] 4.1.1. Analysis of Adipocyte Differentiation Ability

[0147] The cells at passage 7 cultured according to 1.2 (1) and 1.2 (2) of Example 1 were cultured in an adipogenesis differentiation medium (StemPro Adipogenesis Differentiation Kit, Life Technology) for 2 weeks while replacing the medium every three days. Then, the culture medium was removed and cells were washed with Ca/Mg free DPBS, and reacted with 4% paraformaldehyde at room temperature for 15 minutes. The cells were washed with 60% isopropanol and then reacted with Oil Red 0 for 10 minutes. Then, the cells were washed with purified water, and adipocytes were observed under a microscope (FIG. 5).

[0148] 4.1.2. Analysis of Osteocyte Differentiation Ability

[0149] The cells prepared in 1.2 (1) and 1.2 (2) of Example 1 were cultured in an osteogenesis differentiation medium (StemPro Osteogenesis Differentiation Kit, Life Technology) for 2 weeks while replacing the medium every three days. Then, the culture medium was removed and cells were washed with Ca/Mg free DPBS, and reacted with 4% paraformaldehyde at room temperature for 15 minutes. After reaction, the cells were washed with purified water, and reacted with a 1% silver nitrate solution at room temperature for 5 minutes. The cells were washed with purified water and then reacted with a 5% sodium thiosulfate solution at room temperature for 5 minutes. Then, the cells were washed with purified water, and reacted with a 0.1% nuclear fast red solution at room temperature for 5 minutes. Then, the cells were washed with purified water, and calcium deposition sample was observed under a microscope (FIG. 5).

[0150] 4.1.3. Analysis of Chondrocyte Differentiation Ability

[0151] 210.sup.5 of the cells prepared in 1.2 (1) and 1.2 (2) of Example 1 were put in a 15 ml tube, and centrifuged at 1,500 rpm for 5 minutes. The supernatant was discarded, and only cells were cultured in a chondrogenesis differentiation medium (Stem Pro Chondrogenesis Differentiation Kit, Life Technology) with a lid closed loosely for 3 weeks while replacing the medium every three days. Then, the cell mass was made into a paraffin block and cut in a thickness of 2 mm on a slide glass, followed by Alcian blue staining. Thereafter, chondrocytes stained with blue color were analyzed by optical microscope and the results are shown in FIG. 5.

[0152] As shown in FIG. 5, the enhanced postnatal adherent cells of the present disclosure were found to differentiate into adipocytes, osteocytes, or chondrocytes under hypoxia and normoxia conditions.

[0153] 4.2. Characterization According to Growth

[0154] Adherent cells were isolated from the amnion and cultured in the same manner as in the culture method under hypoxia condition of Example 1. To compare doubling times of adherent cells subcultured for 1 passage to 15 passages, each same number of cells was seeded in a 6-well plate, and cells were harvested when they occupied 70%80% of the bottom area of the plate, and the number of cells was measured. The measurement was performed in triplicate. 10 l of a cell suspension was mixed with 10 l of trypan blue, and 10 l thereof was counted by using a hemocytometer. The doubling time, which is a time it takes for a cell to double, was calculated using the total number of cells and the time when the number was measured.

[0155] As shown in FIGS. 8A and 8B, the characteristics were maintained up to about passage 12, and the doubling time of about 20 hours was maintained.

[0156] Further, the adherent cells were cultured to passages 24, and the sizes of the cells were measured. Three cell samples were used, and the sizes of the cells were measured by using an Autocell counter. All of the three adherent cells showed similar growth patterns. As shown in FIG. 8C, the cell size was about 20 m or less until p18, and 13 m14 m at early passages. Meanwhile, as the passage number increased, the cell size increased.

[0157] 4.3. Analysis of Cell Migration Ability and Colony Forming Ability

[0158] To analyze cell's ability to migrate to damaged tissues, a transwell was used to examine cell's migration ability. The upper surface of the transwell was coated with 0.1% gelatin at 37 C. Each 510.sup.5 of the cells cultured under hypoxia and normoxia conditions in Example 1 were suspended in serum-free medium, and seeded to the upper chamber of the transwell insert. PS-CM containing chemokines was added to a culture medium of the lower chamber. Cells were cultured in an incubator overnight. Cells that migrated through the transwell was subjected to giemsa's staining, and the number of cells was counted under an optical microscope to confirm cell migration, and results are shown in FIG. 7.

[0159] It was confirmed that all of the cells cultured under hypoxia and normoxia conditions had migration ability and the migration ability of the adherent cells cultured under the hypoxia condition was improved twice or higher, as compared with that of the cells cultured under the normoxia condition.

[0160] Further, the colony forming ability of the enhanced postnatal adherent cells cultured under normoxia and hypoxia conditions was examined by CFU analysis. To analyze cell's colony forming ability, cells were cultured in a 100 mm culture dish for 10 days to 14 days, and colony formation was examined under a microscope. Next, the cells were washed with DPBS, and then 2 mL to 3 mL of a mixed solution of glutaraldehyde and crystal violet was added to cells in the dish and stained for 30 minutes. The cells were carefully washed with sterile water and the number of colonies was counted under a microscope, and presented as mean values to analyze results. As shown in FIG. 6, colony formation (CFU) of the adherent cells cultured under the hypoxia condition was increased about 1.2 times, as compared with that of the cells cultured under the normoxia condition. In addition, the size of the colony was also increased (FIG. 7B).

Example 5. Examination of Functions of Enhanced Postnatal Adherent Cells

[0161] 5.1. Examination of Immune-Modulating Function

[0162] When monocytes isolated from the peripheral blood are activated with PHA, T cells proliferate. When T cell proliferation is inhibited by co-culturing of the monocytes and the adherent cells of the present disclosure, it may be assured that the adherent cells of the present disclosure have immune-modulating function.

[0163] Monocytes were isolated from the donated peripheral blood by using Ficoll, and used. 50,000 of the cells prepared in Example 1 were seeded in a 24 well plate, and adhered to the plate in an incubator at 37 C. overnight. Thereafter, to activate monocytes stained with CFSE, 10 g/ml of PHA mixed with a culture medium was added. 410.sup.5 cells were added and co-cultured directly or indirectly for 5 days. At this time, the direct co-culture was performed by co-culturing the monocytes and the enhanced postnatal adherent cells in the 24 well, and the indirect co-culture was performed by adding the monocytes in the upper chamber of the Transwell. Thereafter, the monocytes were recovered, and T cell proliferation rates were analyzed by a flow cytometer (FACS Caliber, BD science).

[0164] As a result, it was confirmed that the adherent cells of the present disclosure had the immune-modulating function, and when the adherent cells were co-cultured with monocytes directly or indirectly, all the cells showed the immune-modulating function. Specifically, when analysis was performed according to the donor, the adherent cells derived from all donors showed 40% or more of inhibition rate (FIG. 3). Further, when analysis was performed according to the passage, direct co-culture of the peripheral blood monocytes and the adherent cells showed improved inhibition as the passage increased (FIG. 4).

[0165] 5.2. Analysis of Nerve Regeneration Effect

[0166] A nerve regeneration effect of the enhanced postnatal adherent cells isolated and cultured in Example 1 was analyzed.

[0167] In detail, MEM and conditioned medium of the enhanced postnatal adherent cells were collected and prepared as samples. Thereafter, nerve cells (SH-SY5Y) were inoculated in a 96-well plate. When the cells were proliferated for about 1 day, MEM and the culture medium of the enhanced postnatal adherent cells were added thereto, respectively and cultured for 4 days. A reagent of Cyto X Cell viability assay kit (WST-1) was added to the medium at an amount of 10% thereof, and allowed to react in an incubator for 2 hours to 3 hours. Thereafter, proliferation rates of SH-SY5Y cells were analyzed at 450 nm by using a microreader, and the results are shown in FIG. 13.

[0168] FIG. 13 shows results of analyzing the nerve regeneration effect of enhanced postnatal adherent cells according to a specific embodiment. As shown in FIG. 13, when the proliferation rate of the nerve cells cultured in MEM medium was taken as 100%, the proliferation rate of the nerve cells (represented by ePACs culture medium) cultured in the culture medium of the enhanced postnatal adherent cells (ePACs) was about 280%, which is 2.5 times or higher, as compared with that of the control group.

[0169] The result suggests that the enhanced postnatal adherent cells according to a specific embodiment have the immune disease-improving effect and the nerve regeneration effect, and therefore, the postnatal adherent cells of the present disclosure may be applied to the treatment of neurodegenerative diseases.

Example 6. Comparison of Gene and Protein Expression Characteristics of Enhanced Postnatal Adherent Cells

[0170] 6.1. Comparison of Gene and Protein Expression Between Enhanced Postnatal Adherent Cells and Mesenchymal Stem Cells (BMMSC) at mRNA and Protein Levels

[0171] A difference in gene expression was compared between the placenta-derived adherent cells of the present disclosure and BMMSCs.

[0172] RNAs and proteins were extracted from the enhanced postnatal adherent cells and bone marrow-derived mesenchymal stem cells (BMMSCs) (Available source: Cambridge Univ).

[0173] The extracted total RNAs were isolated and amplified by using a Target Amp-Nano Labeling Kit for an Illumina Expression BeadChip (EPICENTRE, Madison, USA). 500 ng of total RNA and T7 oligo(dT) primer were used to synthesize cDNA, and in vitro transcription was performed by using biotin-UTP to prepare biotin-labeled cRNA. The prepared cRNA was quantified by using a NanoDrop. The prepared cRNA was hybridized onto a HT-12 v4.0 expression beadchip. After hybridization, to remove non-specific hybridization, the DNA chip was washed with a wash buffer of Illumina Gene Expression System (Illumina), and the washed DNA chip was labeled with a fluorescent streptavidin-Cy3 (Amersham). The fluorescent-labeled DNA chip was scanned by using a confocal laser scanner (Illumina) and fluorescent data present in each spot were saved with TIFF image files. TIFF image files were analyzed by BeadStudio version 3 (Illumina) to quantify spot fluorescent intensities. The quantified results were subjected to Gene-Enrichment and functional analysis by using a DAVID (http://david.abcc.ncifcrf.goc/home.jsp) program.

[0174] As a result, when genes showing variances in expression level of 2-fold or more were selected, 2635 genes were observed. Among them, expression levels of 1305 genes were increased and expression levels of 1330 genes were decreased in the postnatal adherent cells of the present disclosure, as compared with BMMSCs. Among them, 23 genes showing the greatest difference in the expression level was selected, and shown in the following Table 5. Among them, COL3A1, IGFBP5, PRNP, MT1A, and CCND1 are genes that showed increased expression levels in the postnatal adherent cells, as compared with BMMSCs. COL1A2, COL1A1, TPM2, TAGLN, CALD1, COL6A3, IGFBP7, SPARC, EFEMP1, CYP1B1, CXCL12 and PENK are genes that showed decreased expression levels in the postnatal adherent cells, as compared with BMMSCs.

TABLE-US-00005 TABLE 5 ProbeID ACCESSION SYMBOL fold change 1 ILMN_1785272 NM_000089.3 COL1A2 2.152358 2 ILMN_1701308 NM_000088.3 COL1A1 2.238551 3 ILMN_1757604 NM_213674.1 TPM2 2.183863 4 ILMN_1773079 NM_000090.3 COL3A1 2.13323 5 ILMN_1778668 NM_003186.3 TAGLN 3.064249 6 ILMN_1803429 NM_001001391.1 CD44 2.548315 7 ILMN_1671703 NM_001613.1 ACTA2 3.620056 8 ILMN_1730487 NM_033140.2 CALD1 2.928108 9 ILMN_1750324 NM_000599.2 IGFBP5 2.981225 10 ILMN_1699829 NM_001901.1 CTGF 3.536551 11 ILMN_1737988 NM_001080121.1 PRNP 2.368955 12 ILMN_1706643 NM_057165.2 COL6A3 2.367989 13 ILMN_1665865 NM_001552.2 IGFBP4 3.947149 14 ILMN_1691156 NM_005946.2 MT1A 2.091371 15 ILMN_2360710 NM_001018004.1 TPM1 4.322069 16 ILMN_2062468 NM_001553.1 IGFBP7 2.435926 17 ILMN_1796734 NM_003118.2 SPARC 2.117699 18 ILMN_1686116 NM_003246.2 THBS1 5.42393 19 ILMN_1688480 NM_053056.2 CCND1 2.499045 20 ILMN_2350634 NM_001039348.1 EFEMP1 2.639375 21 ILMN_1693338 NM_000104.2 CYP1B1 21.273743 22 ILMN_1791447 NM_199168.2 CXCL12 109.091657 23 ILMN_1726711 NM_006211.2 PENK 167.876141

[0175] The extracted total proteins were digested into peptides by FASP which is a tryptic digestion method of using a filter, and for relative quantification, the peptides were subjected to isotope labeling, and mass spectrometry was performed by using an Easy nLC-1000/Q-Exactive system for LC-MS spectrometry.

[0176] As shown in FIG. 12A, when protein expression was compared between the enhanced postnatal adherent cells and BMMSCs, the postnatal adherent cells showed high expression of LIN28B, FERMT3, RAB27B, and PEG10 and low expression of HYOU1 and GLIPR1, as compared with BMMSCs.

[0177] 6.2. Comparison of Gene and Protein Expression Between Enhanced Postnatal Adherent Cells Cultured Under Hypoxia Condition and Under Normoxia Condition at mRNA and Protein Levels

[0178] A difference in gene expression was compared between the adherent cells cultured under a hypoxia condition and the adherent cells cultured under a normoxia condition.

[0179] For comparison, a microarray and a proteomics array were used and a detailed method is the same as in Example 6.1. Further, the enhanced postnatal adherent cells were isolated and cultured in the same manner as in Example 1.

[0180] As a result, when genes showing variances in expression level of 2-fold or more were selected, 514 genes were observed. Among them, expression levels of 229 genes were increased and expression levels of 285 genes were decreased in the postnatal adherent cells under the hypoxia condition. Among them, 21 genes showing the greatest difference in the expression level was selected, and shown in the following Table 6. Among them, PGK1, BNIP3, TPI1, ERRFI1, LOC644774, SLC2A3, PLIN2, and TUBB2B were increased and ALDH1A3, SERPINE1, IL6, AKR1B1, NQO1, PAPPA, TAGLN, TGM2, IL1B, IL8, and EFEMP1 were decreased under the hypoxia condition.

TABLE-US-00006 TABLE 6 Probe ID ACCESSION SYMBOL fold change 1 ILMN_1755749 NM_000291.2 PGK1 2.191413 2 ILMN_1724658 NM_004052.2 BNIP3 2.799374 3 ILMN_1707627 NM_000365.4 TPI1 2.075759 4 ILMN_1665510 NM_018948.2 ERRFI1 3.446231 5 ILMN_2139970 NM_000693.1 ALDH1A3 2.278008 6 ILMN_1744381 NM_000602.1 SERPINE1 2.722234 7 ILMN_1699651 NM_000600.1 IL6 2.278956 8 ILMN_1701731 NM_001628.2 AKR1B1 2.365849 9 ILMN_1779258 XM_927868.1 LOC644774 3.295901 10 ILMN_1720282 NM_000903.2 NQO1 2.35445 11 ILMN_1721770 NM_002581.3 PAPPA 2.210303 12 ILMN_1778668 NM_003186.3 TAGLN 2.595073 13 ILMN_1705750 NM_004613.2 TGM2 2.291753 14 ILMN_1775708 NM_006931.1 SLC2A3 3.072693 15 ILMN_1775501 NM_000576.2 IL1B 2.134024 16 ILMN_2138765 NM_001122.2 PLIN2 2.763918 17 ILMN_2184373 NM_000584.2 IL8 4.543316 18 ILMN_1881909 BU536065 2.996621 19 ILMN_2350634 NM_001039348.1 EFEMP1 2.33499 20 ILMN_1807439 NM_000693.2 ALDH1A3 2.783309 21 ILMN_1680874 NM_178012.3 TUBB2B 4.767344

[0181] As shown in FIG. 12, when protein expression was compared between the postnatal adherent cells cultured under the hypoxia condition and the postnatal adherent cells cultured under the normoxia condition, the postnatal adherent cells cultured under the hypoxia condition showed high expression of PODXL, HIST1H1B, and PLEK2 and low expression of DPP4, as compared with the postnatal adherent cells cultured under the normoxia condition.