WO2017026838A1 - Improved umbilical cord-derived adhesive stem cells, preparation method therefor, and use thereof - Google Patents

Abstract

Disclosed are improved umbilical cord-derived adhesive stem cells, a preparation method therefor, and a use thereof. The improved umbilical cord-derived adhesive stem cells have an anti-inflammatory effect, a blood vessel regeneration effect, or a nerve regeneration effect, thereby being usable in a pharmaceutical composition or a cell therapeutic agent for treating or preventing various diseases.

Description

Improved Umbilical Cord-derived Attached Stem Cells

Improved umbilical cord-derived adherent stem cells, methods of making and uses thereof.

Cell therapy is a medicine used for the purpose of preventing or treating certain diseases by changing the characteristics of cells by proliferating or selecting cells in vitro to restore the function of cells and tissues. The field is receiving. Depending on the degree of differentiation can be divided into somatic and stem cell therapy, stem cell therapy can be divided into embryonic stem and adult stem cells.

Until now, most adult stem cell research has been conducted in bone marrow, and in some cases, stem cells are isolated and cultured from adipose tissue or cord blood. However, bone marrow and adipose tissue cells are collected by invasive methods, and stem cells isolated from mature or old age patients have differentiation and proliferation ability. In addition, in the case of umbilical cord blood collection is easy but the content of stem cells in the umbilical cord blood is low.

The umbilical cord (UC), unlike bone marrow or adipose-derived cells, is extracted from tissues that have already been separated from the body and thus is non-invasive and facilitates the extraction process. In addition, unlike embryonic stem cells, they are free from ethical aspects. Therefore, it has recently been in the spotlight as a useful material of refractory or regenerative medicine, and as a primitive cell, it can satisfy both proliferative capacity and differentiation capacity, and has the advantage that it can be used after differentiation according to organ characteristics as well as organ regeneration. However, since there are many types of cells inside the umbilical cord, studies to find the optimal cell as a therapeutic agent and to identify new characteristics of cells that can be separated or extracted into a uniform cell population are required.

One aspect is to provide an improved umbilical cord derived adherent stem cell or cell population thereof.

Another aspect includes culturing by attaching the separated umbilical cord to the culture vessel; Separating the improved umbilical cord-derived adherent stem cells by contacting the cultured umbilical cord with a separation enzyme; Providing a method for producing an improved umbilical cord-derived stem cells comprising the step of passage the separated improved umbilical cord-derived stem cells in a medium containing fibroblast growth factor-4 (FGF-4) and heparin will be.

Another aspect is to provide a pharmaceutical composition comprising the improved umbilical cord-derived adherent stem cells, their cell populations or their cultures as an active ingredient.

One aspect provides improved umbilical cord derived stem cells.

The improved umbilical cord-derived attached stem cells may be one or more of the following characteristics selected from (a) to (e):

a) at least one selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17 and IL33 is more expressed compared to bone marrow stem cells;

b) one or more selected from the group consisting of CCND1, SERPINE1, PRNP, and CYP1B1 are less expressed than bone marrow stem cells;

c) maintaining the shape of adherent fibroblasts during passaging;

d) ability to differentiate into adipocytes, osteocytes, or chondrocytes; And

e) selected from the group consisting of CD200 +, Tra-1-60-, CD3-, CD1a-, CD11c-, CD16-, CD86-, CD8a-, CD40-, CD141 +, CD61 +, CD87 +, MIC A / B- and SSEA4 + One or more surface antigen properties.

The improved umbilical cord-derived attached stem cells may further have one or more properties selected from the following (f) to (i):

f) at least one selected from the group consisting of S100A10, BNIP3, IGFBP5, NDUFA4L2, DPYD and SCARA3 is expressed more than in culture under normal oxygen conditions;

g) one or more selected from the group consisting of IL8, ALDH1A1, DLC1, CTHRC1, and CPA4 is less expressed compared to culture in normal oxygen conditions;

h) one or more selected from the group consisting of SNCA, DSG2, NRP2, and PLAT is more expressed compared to bone marrow stem cells;

i) one or more selected from the group consisting of TPMT, NAGK, and ANXA4 is less expressed compared to bone marrow stem cells.

The improved umbilical cord-derived attached stem cells may be one of the surface antigen characteristics of e) additionally Oct4-, or Nanog-. In addition, among the surface antigen properties of e), CD61 + may be a surface antigen property overexpressed in hypoxic conditions.

As used herein, the term "umbilical cord" may refer to a line that connects the mother and the abdomen to allow the mammalian fetus to grow in the placenta, generally three vessels surrounded by Wharton jelly, ie two umbilical arteries It may mean a tissue consisting of and one umbilical vein. Thus, the term "enhanced Umbilical Cord Adherent Stem cells" or "Umbilical Cord Adherent Stem cells" herein refers to wharton's Jelly tissue of the umbilical cord or umbilical cord. It can mean a cell derived from, having the ability to differentiate into a variety of tissue cells and has the property of growing on the surface of the culture vessel.

The improved umbilical cord-derived adherent stem cells provided herein provide for at least about 20%, 25%, 30%, 35%, CD200, CD141, CD61, CD87, or SSEA4 positive surface markers for cell markers expressed on the cell surface. 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or about 99% and expresses stem cell marker Oct4 At least about 70%, at least 60%, at least 50%, at least 40%, at least Nanog, Tra-1-60, CD3, CD1a, CD11c, CD16, CD86, CD8a, MIC A / B or CD40 negative markers , At least 30% or less, at least 20% or less, at least 10% or less, at least 5% or less, or at least 1% or less. As used herein, the term "positive" may refer to a stem cell label, which means that the label is present in a greater amount, or at a higher concentration as compared to other non-stem cells on which it is a reference. That is, a cell is positive for that label because a label is present inside or on the surface of the cell and the label can be used to distinguish the cell from one or more other cell types. It may also mean that the cell has its label in an amount sufficient to give a signal greater than the background value, for example a signal from a cytometry device. For example, if a cell can be detectably labeled with an antibody specific for CD200 and the signal from this antibody is detectably greater than the control (eg background value), then the cell is "CD200 +". As used herein, the term “negative” means that even when an antibody specific for a specific cell surface label is used, the label cannot be detected in comparison with the background value. For example, if a cell cannot be detectably labeled with an antibody specific for CD3, the cell is "CD3-".

Such immunological properties can be determined by conventional methods known in the art. For example, various methods may be used, such as flow cytometry, immunohistochemical staining, or RT-PCR.

An improved umbilical cord-derived adherent stem cell according to one embodiment may express more than one gene or protein selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17 and IL33 compared to bone marrow-derived stem cells. Specifically, at least two or three or more genes or proteins selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17 and IL33 in the cells, or more specifically, all genes or proteins are more than bone marrow-derived stem cells. Can be expressed. Compared to the bone marrow-derived stem cells, the genes expressed more in the improved umbilical cord-derived adherent stem cells according to one embodiment more S100A10, SQSTM1, DSTN, DCN, PHGDH, FBLN1, MFGE8, HLA-A, VASN, or KIAA1199 It may include. The gene has not been reported for its association with enhanced umbilical cord derived stem cells. The improved umbilical cord-derived stem cells according to one embodiment may exhibit a difference in expression levels of two or more times for the gene as compared to the bone marrow-derived stem cells. The difference in the expression level may be, for example, comparing the expression level of the gene at the mRNA level. The expression level difference may also be, for example, by microarray analysis.

In addition, the improved umbilical cord-derived attached stem cells according to one embodiment may express less than one or more genes or proteins selected from the group consisting of CCND1, SERPINE1, PRNP, and CYP1B1 compared to bone marrow-derived stem cells. Specifically, at least two, or at least three, genes or proteins selected from the group consisting of CCND1, SERPINE1, PRNP, and CYP1B1 in the cells, or more specifically, all genes or proteins may be less expressed than bone marrow-derived stem cells. . Less gene or protein expressed in the improved umbilical cord-derived attached stem cells according to one embodiment compared to the bone marrow-derived stem cells may include MTA2A, TM4SF1, HIST1H4C, and NME1. The gene or protein has not been reported for its association with improved umbilical cord derived stem cells. The improved umbilical cord-derived stem cells according to one embodiment may exhibit a difference in expression levels of two or more times for the genes or proteins as compared to the bone marrow-derived stem cells. The difference in the expression level may be, for example, comparing the expression level of the gene at the mRNA level. The expression level difference may also be, for example, by microarray analysis.

In addition, the improved umbilical cord-derived stem cells may have the form of subcultured fibroblasts. In one embodiment, the cell may have a property of a cell that requires attachment to a surface for growth in vitro, and may exhibit a specific form of fusiform fibroblasts.

In another embodiment, the improved umbilical cord-derived attached stem cells may be one having colony forming ability. The cells may have a high colony forming ability as compared to culture in normal acid conditions.

In addition, the improved umbilical cord-derived stem cells may be differentiated into adipocytes, osteocytes or chondrocytes. The cells can be induced to differentiate along specific cell lineages, including, for example, adipocyte differentiation, chondrocyte differentiation, osteoblast differentiation, hematopoietic cell differentiation, myocyte differentiation, vascular cell differentiation, neuronal differentiation, hepatocyte differentiation.

The term "differentiation" refers to a phenomenon in which structures or functions are specialized to each other during cell division, proliferation, and growth, that is, changes in form or function to perform a task given to each cell, tissue, etc. of an organism. Measurement of differentiation into specific cell types can be performed by methods well known in the art, and can induce differentiation into specific cells through known methods. The differentiation may also be performed using techniques such as flow cytometry or immunocytochemistry to measure cell surface labeling (e.g., staining cells with tissue-specific or cell-labeling specific antibodies) and morphological changes, Or by examining the morphology of the cells using confocal microscopy, or by measuring changes in gene expression using techniques well known in the art such as PCR and gene-expression profiles.

In addition, the improved umbilical cord-derived stem cells are IL-6, IL-8, G-CSF, GM-CSF, MCP-3, VEGF, GRO, IFNγ, IL-1a, IL-1b, IL-1ra, IL- 3, IL-4, IL-7, IL-9, IL-12 (p40), IL12 (P70), IL-13, IL-14, IFNα2, MDC, sIL-2Ra, Eotaxin, Flt-3 ligand, MCP Can secrete proteins of −1, MIP-1a, MIP1b, RANTE, Fractalkine, IP-10, EGF, FGF-2, IGF-1 SR, EpCAM, IGFBP3 or a combination thereof. Also for example, the cell is at least two, at least three, at least four, five of the protein selected from the group consisting of IL-6, IL-8, G-CSF, GM-CSF, MCP-3, VEGF, and GRO At least 6, at least 7, at least 8, at least 9, at least 10 or all proteins can be secreted.

In addition, the improved umbilical cord-derived stem cells are S100A10, BNIP3, IGFBP5, PGK1, TPI1, DCN, PGM1, PFKFB3, LOC644774, MME, MIR1978, SLC2A3, BHLHB2, in cells cultured in hypoxic conditions compared to normal oxygen culture conditions. The expression level of one or more genes or proteins selected from the group consisting of BNIP3L, IGFBP5, NDUFA4L2, DPYD, and SCARA3 may be increased. Specifically, the improved umbilical cord-derived stem cells cultured in hypoxic culture conditions were S100A10, BNIP3, IGFBP5, PGK1, TPI1, DCN, PGM1, PFKFB3, LOC644774, MME than the improved umbilical cord-derived stem cells cultured in normal oxygen culture conditions. Can increase the expression level of at least 2, at least 3, at least 4, at least 5, at least 6, or all genes or proteins selected from the group consisting of MIR1978, SLC2A3, BHLHB2, BNIP3L, IGFBP5, NDUFA4L2, DPYD, and SCARA3. have. The gene or protein has not been reported for its association with improved umbilical cord derived stem cells. The difference in expression levels can be two or more times. The difference in expression level may be, for example, a comparison of expression levels of genes and proteins at the mRNA or protein level. The expression level difference can also be, for example, by microarray and proteomics analysis.

In addition, the improved umbilical cord-derived attached stem cells have an expression level of one or more genes or proteins selected from the group consisting of IL8, ALDH1A1, NQO1, DLC1, CTHRC1 and CPA4 in cells cultured in hypoxic culture conditions compared to normal oxygen culture conditions. May be decreasing. Specifically, the improved umbilical cord-derived stem cells cultured in hypoxic culture conditions are at least two selected from the group consisting of IL8, ALDH1A1, NQO1, DLC1, CTHRC1 and CPA4 than the improved umbilical cord-derived stem cells cultured in normal oxygen culture conditions. The level of expression of three or more or all genes or proteins may decrease. The gene or protein has not been reported for its association with improved umbilical cord derived stem cells. The difference in expression levels can be two or more times. The difference in expression level may be, for example, a comparison of expression levels of genes and proteins at the mRNA or protein level. The expression level difference can also be, for example, by microarray and proteomics analysis.

Another aspect provides an improved umbilical cord derived stem cell population.

The umbilical cord-derived attached stem cells are as described above.

Another aspect includes culturing by attaching the separated umbilical cord to the culture vessel; Separating the improved umbilical cord-derived adherent stem cells by contacting the cultured umbilical cord with a separation enzyme; It provides a method for producing an improved umbilical cord-derived attached stem cells comprising the step of passage the separated improved umbilical cord-derived attached stem cells in a medium containing fibroblast growth factor-4 (FGF-4) and heparin. .

The umbilical cord may use a placenta isolated after childbirth from a healthy mother (eg, HIV, HCV, HBV negative mother). That is, the term "isolated umbilical cord" may refer to an umbilical cord separated after giving birth from a mother's mother. The separated umbilical cord may be stored in a sterilized container and ice quickly after being separated.

The method of obtaining separating the umbilical cord from the placenta includes, for example, separating the umbilical cord from the separated placenta; Removing blood outside the separated umbilical cord; Removing the arteries and veins of the umbilical cord from which the blood is removed; And / or severing the blood from which the arteries and veins have been removed to a predetermined size (eg, 1 to 20 mm). For the removal of the blood, for example, Ca / Mg free DPBS, or gentamicin-containing Ca / Mg free DPBS can be used.

Next, the step of separating the stem cells from the fragmented umbilical cord (eg, separated umbilical cord) may be performed. Separating the improved umbilical cord-derived attached stem cells may be attached to the cultured umbilical cord attached to the culture vessel for 5 to 20 days, for example, 10 to 20 days, for example, 10 to 15 days; Confirming that the cells extend from the cultured umbilical cord tissue; And / or treating the sequestration enzyme in the umbilical cord tissue.

The separating enzyme may include collagenase. The collagenase may refer to an enzyme that breaks down the peptide bonds of collagen, and may include collagenase type I, type II, type III, type IV, or a combination thereof. The degrading enzyme may comprise 5 to 30 U / ml, for example 5 to 25 U / ml, 10 to 25 U / ml, or 20 U / ml of collagenase. In addition, the separation enzyme may include trypsin, and / or dispase, and the solution containing the separation enzyme may also include collagenase, trypsin, and / or dispase. Water, saline, for example, HBSS (Hank's Balanced Salt Solution) may be included. In addition, the treatment time of the separation enzyme may be, for example, 1 hour to 20 hours, 2 hours to 10 hours, 4 hours to 9 hours, or 5 hours to 6 hours.

In one embodiment, the reaction of the tissue and the separation enzyme may be a reaction by performing shaking, the shaking is about 20 to 40 ℃, about 30 to 40 ℃, or 35 to 40 ℃, for example, 37 It may be carried out at ℃, may be carried out for about 5 to 60 minutes or 10 to 30 minutes, it may also be carried out twice, for example 10 to 30 minutes.

In addition, after the reaction between the tissue and the separation enzyme, a process for inactivating the separation enzyme may be further performed. For example, FBS may be added to stop the enzyme reaction. In addition, the method for separating tissue cells, for example, improved umbilical cord-derived stem cells from the enzyme reaction solution can be carried out by a method known in the art, for example, after centrifugation, the cell body ( cells can be isolated using a strainer.

As used herein, the term "isolation of improved umbilical cord-derived adherent stem cells" refers to at least 20%, 30%, 40%, 50%, 60%, 70% of cells normally associated with stem cells in an untreated mammalian umbilical cord. May mean removing 80%, 90%, 95% or 99%. A population of cells containing stem cells from one organ can be said to be "isolated" when the other cell that is normally associated with the stem cells in the organ is untreated.

 Next, the isolated improved umbilical cord-derived attached stem cells may include a step of passage culture with P0.

The passaging step may further include the treatment of an Animal Component Free (ACF) recombinant enzyme prior to cell transplantation for passaging. As used herein, the term "Animal Component Free Enzyme" is of non-animal origin, which may mean that the enzyme is not purified from an animal source. The enzyme without the animal derived component may be of recombinant origin, for example bacterial, yeast or plant origin. Enzymes of recombinant origin may refer to any enzyme produced by recombinant DNA technology, including the use of microorganisms such as bacteria, viruses, yeast, plants, etc. for their production. The enzyme can be recombinant trypsin without animal derived components, for example recombinant trypsin produced in corn. Recombinant trypsin without the animal-derived component is commercially available, for example TrypLE ^™ Select (GIBCO Invitrogen), TrypLE ^™ Express (GIBCO Invitrogen), TrypZean ^™ (Sigma Aldrich) or Recombinant Trypsin Solution ^™ (Biological Industries) Can be.

The passaging step includes culturing in a stem cell culture medium, for example, fibroblast growth factor-4 (FGF-4) and heparin added medium. FGF-4 in the medium may be added at a concentration of about 10 ng / ml to about 40 ng / ml, or about 20 ng / ml to about 30 mg / ml, for example 25 ng / ml. Heparin in the medium may be added at a concentration of about 0.5 μg / ml to 2 μg / ml, or 0.5 μg / ml to 1.5 μg / ml, for example 1 μg / ml. The medium may further include, for example, fetal bovine serum, and antibiotics (eg, penicillin, streptomycin, gentamicin, etc.). In one embodiment, CS-CM medium with 10% fetal bovine serum, 50 μg / ml gentamicin, 1 μg / ml heparin, and 25 ng / ml FGF-4 added can be used. The passage culture may be performed at about 20 to 40 ℃, about 30 to 40 ℃, or 35 to 40 ℃, for example, 37 ℃, the incubation time for each passage is, for example, 2 to 7 days, Or 3 to 5 days.

In the improved method of producing umbilical cord-derived adherent stem cells according to one embodiment, the passage number of the passage is not particularly limited, and may be appropriately selected according to the number of desired proliferating cells. Typically, at least one passage or more than 10 passages may be used. For example, by performing 1 to 20 passages and 3 to 15 passages, a clinically necessary number of cumulative proliferating cells can be obtained.

In addition, in the subculture, as described above, the treatment of a recombinant enzyme without an animal-derived component may be additionally performed. That is, the purity of the cells can be improved by collecting the cells by treating the enzymes without animal-derived components before passing the cells to the next step for each passaging step. For example, in a step from P1 to P2, the recombinant enzyme without the animal-derived component may be treated before transplanting the cells for P2.

The passaging step may be to passaging under low oxygen conditions compared to the normal oxygen conditions 21%. The term "hypoxia" may refer to a low oxygen partial pressure condition compared to 21% oxygen partial pressure, which is a normal normal oxygen condition. The low oxygen condition may be a state having an oxygen partial pressure of 1 to 15%, 1 to 12%, 1 to 10%, or 1 to 5%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%.

In one embodiment, S100A10, BNIP3, IGFBP5, PGK1, TPI1, DCN, PGM1, PFKFB3, LOC644774, MME, MIR1978, SLC2A3, BHLHB2, BNIP3L, IGFBP5, NDUFA compared to normal oxygen conditions in passage under hypoxic conditions The expression of any one or more selected from the group consisting of DPYD and SCARA3 may be increased or the expression of any one or more selected from the group consisting of IL8, ALDH1A1, NQO1, DLC1, CTHRC1 and CPA4 may be decreased.

The improved umbilical cord-derived stem cells produced by the production method have the characteristics as described above. For example, the prepared umbilical cord-derived stem cells may be selected from the following (a) to (e). It may have the following characteristics:

a) at least one selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17 and IL33 is more expressed compared to bone marrow stem cells;

b) one or more selected from the group consisting of CCND1, SERPINE1, PRNP and CYP1B1 are less expressed than bone marrow stem cells;

c) maintaining the shape of adherent fibroblasts during passaging;

d) ability to differentiate into adipocytes, osteocytes, or chondrocytes; And

e) selected from the group consisting of CD200 +, Tra-1-60-, CD3-, CD1a-, CD11c-, CD16-, CD86-, CD8a-, CD40-, CD141 +, CD61 +, CD87 +, MIC A / B- and SSEA4 + One or more surface antigen properties.

The improved umbilical cord-derived attached stem cells may further have one or more properties selected from the following (f) to (i):

f) at least one selected from the group consisting of S100A10, BNIP3, IGFBP5, NDUFA4L2, DPYD and SCARA3 is expressed more than in culture under normal oxygen conditions;

g) one or more selected from the group consisting of IL8, ALDH1A1, DLC1, CTHRC1, and CPA4 is less expressed compared to culture in normal oxygen conditions;

h) one or more selected from the group consisting of SNCA, DSG2, NRP2, and PLAT is more expressed compared to bone marrow stem cells;

i) one or more selected from the group consisting of TPMT, NAGK, and ANXA4 is less expressed compared to bone marrow stem cells.

The improved umbilical cord-derived attached stem cells may be one of the surface antigen characteristics of e) additionally Oct4-, or Nanog-. In addition, among the surface antigen properties of e), CD61 + may be a surface antigen property overexpressed in hypoxic conditions.

Another aspect is to provide a cell therapy, pharmaceutical composition or formulation comprising the improved umbilical cord-derived adherent stem cells, their cell populations or their cultures as an active ingredient.

Another aspect provides the use of said improved umbilical cord-derived adherent stem cells, populations thereof or cultures thereof for use in the manufacture of cell therapies, pharmaceutical compositions or formulations.

For example, an improved umbilical cord-derived adherent stem cell having one or more of the following properties selected from (a) to (e), a cell therapy, pharmaceutical composition, or agent comprising a cell population thereof may be provided:

a) at least one selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17 and IL33 is more expressed compared to bone marrow stem cells;

b) one or more selected from the group consisting of CCND1, SERPINE1, PRNP and CYP1B1 are less expressed than bone marrow stem cells;

c) maintaining the shape of adherent fibroblasts during passaging;

d) ability to differentiate into adipocytes, osteocytes, or chondrocytes; And

e) selected from the group consisting of CD200 +, Tra-1-60-, CD3-, CD1a-, CD11c-, CD16-, CD86-, CD8a-, CD40-, CD141 +, CD61 +, CD87 +, MIC A / B- and SSEA4 + One or more surface antigen properties.

The improved umbilical cord-derived attached stem cells may further have one or more properties selected from the following (f) to (i):

f) at least one selected from the group consisting of S100A10, BNIP3, IGFBP5, NDUFA4L2, DPYD and SCARA3 is expressed more than in culture under normal oxygen conditions;

g) one or more selected from the group consisting of IL8, ALDH1A1, DLC1, CTHRC1, and CPA4 is less expressed compared to culture in normal oxygen conditions;

h) one or more selected from the group consisting of SNCA, DSG2, NRP2, and PLAT is more expressed compared to bone marrow stem cells;

i) one or more selected from the group consisting of TPMT, NAGK, and ANXA4 is less expressed compared to bone marrow stem cells.

The aspect also includes a pharmaceutical composition comprising a culture of the improved umbilical cord-derived adherent stem cells. In addition, for example, the pharmaceutical composition for the treatment or prevention of inflammatory diseases, ischemic diseases, and / or neurodegenerative diseases comprising the improved umbilical cord-derived attached stem cells, their cell population, or a culture medium thereof as an active ingredient. to provide.

In addition, another aspect is such improved umbilical cord-derived adherent stem cells for use in the manufacture of a medicament for use in the treatment or prevention of diseases such as inflammatory diseases, ischemic diseases, and / or neurodegenerative diseases, cell populations thereof. Or the use of the culture solution thereof.

Also another aspect is a disease, eg, inflammatory disease, ischemic disease, and / or administration comprising administering the improved umbilical cord-derived attached stem cells, populations thereof, or cultures thereof to an individual in need thereof. Or methods of treating or preventing neurodegenerative diseases.

The improved umbilical cord-derived attached stem cells are as described above.

An improved umbilical cord-derived adherent stem cell according to one embodiment is a protein that is beneficial for treating a disease as described above (eg, IL-6, IL-8, G-CSF, GM-CSF, MCP-3, VEGF, or GRO) and its ability to migrate to damaged tissues are not only remarkable, but also have anti-inflammatory, blood vessel regeneration, and nerve regeneration effects, thus preventing various diseases including inflammatory diseases, ischemic diseases, and / or neurodegenerative diseases. Or may be usefully used in cell therapeutics or pharmaceutical compositions for treatment.

Examples of such diseases may include inflammatory diseases, ischemic diseases, and / or neurodegenerative diseases. Examples of the inflammatory disease may include bronchitis, gastritis, arteriosclerosis, arthritis, inflammatory bowel disease (IBD), hepatitis, cholecystitis, fungal infection, gastric ulcer, asthma, atopic dermatitis, tendinitis or nephritis . Examples of such ischemic diseases include ischemic stroke, myocardial infarction, ischemic heart disease, ischemic brain disease, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, or ischemic leg disease Can be. As used herein, "ischemic stroke" or "stroke" may mean a disease caused by necrosis of brain tissue or cells due to a decrease in cerebral blood flow for a predetermined time, and "cerebral infarction". Can be used interchangeably.

Examples of such neurodegenerative diseases include spinal cord injury, multiple sclerosis, Alzheimer's disease, Frontotemporal dementia, progressive supranuclear palsy, cortical basal degeneration, and Pick's disease. ), Or boxer dementia (Dementia pugilistica, DP).

The dosage of the cell therapy or pharmaceutical composition according to one embodiment is 1.0 X 10 ³ to 1.0 X 10 ¹⁰ cells / kg body weight or individual, or 1.0 X 10 ⁷ to 1.0 X, based on the improved umbilical cord-derived adherent stem cells. 10 ⁸ cells / kg body weight or individual. However, the dosage may be variously prescribed by such factors as the formulation method, the mode of administration, the age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and reaction sensitivity of the patient. These factors can be taken into account to properly adjust the dosage. The number of administrations may be one or two or more times within the range of clinically acceptable side effects, and may be administered to one or two or more sites of administration. Also for animals other than humans, the same dosage as humans per kg or individual, or the above-mentioned administration at the volume ratio (for example, average value) of the organ (heart, etc.) between the target animal and humans, etc. The amount converted into the amount can be administered. Examples of the target animal for the treatment according to one embodiment include humans and mammals for other purposes, and specifically, humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs, and the like. Included.

Cell therapy or pharmaceutical composition according to one embodiment may include the enhanced umbilical cord-derived attached stem cells and a pharmaceutically acceptable carrier and / or additives as an active ingredient. Examples include sterile water, physiological saline, conventional buffers (phosphate, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do. For topical administration, it is also preferable to combine organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof. . When the cell therapy or pharmaceutical composition according to one embodiment is formulated in a formulation suitable for injection, the cell aggregate may be dissolved in a pharmaceutically acceptable carrier or frozen in solution.

Improved umbilical cord-derived adherent stem cells according to one embodiment are various types in which the tissues or organs of the body are fortified, treated or replaced by engraftment, transplantation or infusion of the desired cell population, for example stem cells or derived cell populations. Can be used in the treatment protocol. The improved umbilical cord-derived adherent stem cells may replace or enhance existing tissue, resulting in new or altered tissue or combined with biological tissue or structures. In addition, stem cells may be replaced with the improved umbilical cord-derived stem cells of the present disclosure in treatment protocols where typically tissue-derived stem cells other than the umbilical cord are used.

The cell therapeutic agent or pharmaceutical composition according to one embodiment may be prepared according to the administration method or dosage form thereof, and as necessary, suspending agents, dissolution aids, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, A passivating agent, a buffer, a reducing agent, an antioxidant, etc. can be included suitably. Pharmaceutically acceptable carriers and formulations suitable for the present invention, including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.

The cell therapeutic agent or pharmaceutical composition according to one embodiment is formulated with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by one of ordinary skill in the art. It may be prepared in unit dose form or incorporated into a multi-dose container. The formulations can then be in the form of solutions, suspensions or emulsions in oil or aqueous media or in the form of powders, granules, tablets or capsules. In addition, cell therapy agents may be formulated into injectable formulations. In this case, known conventional ingredients for formulating can be used and can be formulated in conventional manner.

According to an improved umbilical cord-derived adherent stem cell according to one aspect, there is an anti-inflammatory effect, a blood vessel regeneration effect, or a nerve regeneration effect, and thus may be usefully used in pharmaceutical compositions or cell therapeutics for the treatment or prevention of various diseases.

According to the improved method of producing cord-derived adherent stem cells according to another aspect, there is an effect that can increase the purity, yield and proliferation of stem cells from the umbilical cord tissue.

1A is a diagram showing the cell morphology before and after the separation enzyme in improved umbilical cord-derived adherent stem cell separation according to one embodiment.

Figure 1b is a diagram showing the cell morphology according to the treatment time of the separation enzyme in improved umbilical cord-derived adherent stem cell separation according to one embodiment: G1: Col I treatment group, G2: Col I treatment group after tissue attachment.

2 is a diagram illustrating a comparison between hypoxic and normal oxygen conditions in improved umbilical cord-derived attached stem cell culture according to one embodiment: 3%: low oxygen partial pressure (3% O 2), 21%: normal oxygen Partial pressure (21% O2).

3 is a view showing the results of performing karyotype analysis of the genetic stability of the improved umbilical cord-derived stem cells according to one embodiment.

Figure 4 is a view showing the results of analyzing the surface protein of the umbilical cord-derived attached stem cells according to one embodiment.

Figure 5 is a view showing the results of analyzing the multipotency of the umbilical cord-derived attached stem cells according to one embodiment.

Figure 6 is a view showing a result of comparing the analysis of protein expression of the improved umbilical cord-derived stem cells according to one embodiment.

Figure 7a is a view showing the results of analyzing the anti-inflammatory effect of the umbilical cord-derived attached stem cells according to one embodiment

Figure 7b is a view showing the results of analyzing the blood vessel regeneration effect of the improved umbilical cord-derived attached stem cells according to one embodiment

Figure 7c is a view showing the result of analyzing the neuronal regeneration effect of the improved umbilical cord-derived attached stem cells according to one embodiment.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

Example  1: Preparation, Characterization of Enhanced Umbilical Cord-derived Attached Stem Cells, and Analysis of Anti-inflammatory, Neuronal and Vascular Regeneration Effects

1. Comparative Analysis of Advanced Umbilical Cord-derived Attached Stem Cells

(1.1) Improved Isolation and Culture of Umbilical Cord-derived Attached Stem Cells 1

Informed consent was obtained from a healthy mother who had delivered normally and informed consent, and the cord was isolated from placental tissue collected at normal placental delivery. The separated umbilical cord was washed 2 to 5 times with Ca / Mg free DPBS to remove blood. Thereafter, the outer amnion was not peeled off, and two arteries and one vein were removed, and the umbilical cord was cut to a size of 1 to 5 mm. Thereafter, the umbilical cord was attached to the culture vessel and cultured for 10 to 15 days. After confirming that the cells were stretched from the cultured tissue, the treated umbilical cord was treated with 200 U / ml collagenase I for 5 to 6 hours. Derived adherent stem cells were isolated. Before and after the collagenase I treatment, the cell morphology was confirmed at 40x and 100x magnification under an optical microscope in order to confirm that the cells extend from the umbilical cord attached tissues, and the results are shown in FIG. 1A.

1A is a diagram showing the cell morphology before and after the separation enzyme in improved umbilical cord-derived adherent stem cell separation according to one embodiment.

As shown in Figure 1a, it can be seen that after treatment with the collagenase I, a homogenous cell morphology.

Subsequently, the separated cells were P0, and were treated with MEM alpha GlutaMAX (CS-CM medium) containing 25 ng / ml FGF4, 1 ug / ml heparin, and 10% FBS at 37 ° C. under hypoxic culture conditions (O ₂ 3%). Incubated. Every 3 to 4 days thereafter, the CS-CM medium was replaced to remove cells that did not adhere to the bottom of the flask. (3 min) treatment and subculture.

(1.2) Improved Isolation and Culture of Umbilical Cord-derived Attached Stem Cells 2

The umbilical cord-derived attached stem cells were isolated and cultured in the same manner as in (1.1) except that the treatment of collagenase I in (1.1) was performed before attaching the umbilical cord to the culture vessel.

(1.3) Improved Isolation and Culture of Umbilical Cord-derived Attached Stem Cells 3

Except that the stem cells isolated in (1.1) were performed under normal oxygen conditions (O ₂ 21%), the improved cord-derived attached stem cells were isolated and cultured in the same manner as in (1.1).

(1.4) Comparative analysis according to the time of separation enzyme treatment

In order to compare the cell recovery rate of the stem cells separated in (1.1) and (1.2), the stem cells isolated in (1.2) and the stem cell group isolated in (1.1) are named G1 and G2, respectively, The morphology of the cells was confirmed at 40 × and 100 × magnification under the light microscope, and the results are shown in FIG. 1B.

Figure 1b is a diagram showing the cell morphology according to the treatment time of the separation enzyme in the improved umbilical cord-derived adherent stem cell separation according to one embodiment.

In addition, the tissue weight of the G1 and G2, the number of cells after treatment with the enzyme and cell number (P0) was compared.

Military composition		Tissue weight (g)	Cell number after separation enzyme treatment	Cell number (P0)
G1	Col I treatment group	2.71	3.1 X 10 5	3.02 X 10 5
G2	Col I treatment group after tissue attachment	3.14	-	3.45 X 10 5

As shown in FIG. 1b and Table 1, when the separation enzyme is treated before the umbilical cord culture, most of the pebble forms, and the proliferation is slow, the cell yield is low, but homogeneous cells when the separation enzyme is treated after the umbilical cord culture It appears that the form of, the proliferation is fast, it can be confirmed that the cell yield is high.

(1.5) Comparative analysis of hypoxic and normal oxygen conditions

To compare the growth curve and the doubling time of adherent cells 1 to 20 passages (P1 to P20) under hypoxic conditions of (1.1) and normal oxygen conditions of (1.2), 6 wells were respectively Cells were collected when the plate was inoculated to account for 70-80% of the plate bottom area. Thereafter, 10 µl of a mixture of 10 µl of sample and 10 µl of trypan blue reagent was injected into one measuring unit of the hemocytometer. Cell counts were measured using an automatic cell counter. At this time, the time was also recorded and the doubling time was calculated. Doubling time was calculated by using the total number of cells and the time of measuring the time to double the number of cells, the results are shown in Figures 2a and 2b.

In addition, to analyze the colony forming ability of the cells, 150 cells per dish were applied to 100 mm culture dishes. Thereafter, after 10 to 14 days of culture in 12 ml culture medium, the presence of cell colonies was confirmed under a microscope. Next, after washing with DPBS, 2 to 3 ml of glutaaldehyde and crystal violet mixed solution was added to a dish containing cells and stained for 30 minutes. Carefully washed with sterile water and the number of colonies under the microscope was counted and the average value was analyzed to analyze the results, the results are shown in Figure 2c.

In addition, 0.1% gelatin was coated on the upper surface of the transwell filter for 1 hour at 37 ° C to analyze the ability of cells to migrate to damaged tissues. 5 to x 10 ⁵ cells suspended in serum-free medium of 100 ㎕ was added to the upper transwell chamber, the insertion portion. Starvation overnight in serum free medium prior to addition. 600 μl of the medium containing the chemostat factor (CS-CM) was added to the lower chamber. The cells were then incubated overnight in the incubator. The upper side of the filter was carefully washed with cold PBS and then a swab was used to remove the cells remaining on the upper side of the filter. The transwell filter was cut with a dissecting knife and stained with gold porcelain and placed on a slide glass with the bottom face up. The transferred cells were counted the number of stained cells using an optical microscope, the results are shown in Figure 2d.

2 is a diagram illustrating a comparison between hypoxic conditions and normal oxygen conditions in an improved umbilical cord-derived attached stem cell culture according to one embodiment.

As shown in Figures 2a and 2b, it can be seen that under low oxygen conditions, the cells increase in proliferation rate by shortening the doubling time. In addition, as shown in Figure 2c, when the number of colonization formation increased about two times, as shown in Figure 2d, it can be seen that the ability of the cell to move to the damaged tissue is about 1.6 times improved.

2. Characterization of Advanced Umbilical Cord Derived Stem Cells

(2.1) Genetic safety analysis of improved umbilical cord-derived stem cells

GTG-Banding assay was performed to analyze the genetic safety of the improved umbilical cord-derived stem cells prepared in (1.1) and (1.3).

Specifically, DNA was extracted from P7 and P14 cells using a Promega DNA Extraction Kit and used as a sample. An Illumina HumanOmni1-Quad Chip was used and measured using an iSCAN® scanner. First, 400 ng of each DNA sample is amplified by whole genome amplification, randomly fragmented by chemical method, purified by 2-propanol precipitation, and the chip is buffered before loading the DNA sample. DNA samples were added to the chips pretreated with the solution. Following incubation for about 16 hours, staining, allele specific primer extension (ASPE), hybridization, target removal, and washing were performed. Then, scanning was performed with IlluminaScan, and the data was analyzed using GenomeStudio® software, and the results are shown in FIG. 3.

3 is a view showing the results of performing karyotype analysis of the genetic stability of the improved umbilical cord-derived stem cells according to one embodiment.

As shown in Figure 3, the improved umbilical cord-derived attached stem cells produced by the manufacturing method according to one embodiment can be seen that no genetic variation occurs until P14.

(2.2) Surface Protein Analysis of Advanced Umbilical Cord-derived Attached Stem Cells

Flow cytometry and immunofluorescence staining analysis were performed for surface protein analysis of the improved umbilical cord-derived stem cells prepared in (1.1).

Specifically, for flow cytometry, the cells were washed using DPBS, and then placed in DPBS containing 2% FBS, followed by Tra-1-60, CD3, CD1a, CD11c, CD16, CD14, CD86, CD8a, CD19, CD40, CD80. , CD200, CD141, CD61, CD87, MIC A / B, SSEA4 markers were reacted for 20 minutes on ice. Then, the surface antigen was analyzed by flow cytometry (FACS Calibur, Becton Bickinson), the results are shown in Figure 4a.

Immunofluorescence staining was performed to confirm the expression of embryonic stem cell markers Oct4 and Nanog. First, the cells were washed three times with DPBS, and then 4% paraformaldehyde was added to each culture vessel and fixed at room temperature for 10 minutes. The fixed cells were washed three times with DPBS. Next, 0.2% Triton X-100 solution was added and infiltrated at room temperature for 10 minutes, and then washed three times with DPBS. 10% normal goat serum was added and blocked for 30 minutes at room temperature. After the addition of primary antibodies (Oct4, Nanog), light was blocked and reacted at 4 ° C. overnight. Then, after washing three times with DPBS, the secondary antibody was added and reacted at room temperature for 1 hour. Finally, after washing three times with DPBS and observed under a fluorescence microscope, the results are shown in Figure 4b.

Figure 4 is a view showing the results of analyzing the surface protein of the umbilical cord-derived attached stem cells according to one embodiment.

As shown in Figure 4a, improved umbilical cord-derived adherent stem cells according to one embodiment is a cell selectively positive for CD200, CD141, CD61, CD87 SSEA4, TRA-1, CD3, CD1a, CD11c, CD16, CD86 , CD8a, CD40, and MIC A / B cells that are selectively negative, and in addition, CD61 is a cell selectively positive in hypoxic conditions. In addition, as shown in Figure 4b, the improved umbilical cord-derived attached stem cells according to one embodiment does not express the embryonic stem cell specific markers Oct4, Nanog proteins.

(2.3) Enhanced Differentiation of Umbilical Cord-derived Attached Stem Cells

(2.3.1) Adipocyte Differentiation Capacity Analysis

Adipocyte differentiation capacity analysis of the improved umbilical cord-derived adherent stem cells was performed by the following method.

The improved umbilical cord-derived stem cells prepared in (1.1) and (1.3) above were put into Adipogenesis differentiation media (StemPro® Adipogenesis Differentiation Kit, Life Technology), and the medium was exchanged every three days for two weeks. While culturing. After that, the culture solution was removed, washed with Ca / Mg free DPBS, 4% paraformaldehyde was added and reacted at room temperature for 15 minutes. After washing with 60% isopropanol and then adding Oil Red O and reacting for 10 minutes, washing with purified water and observing fat cells under a microscope, the results are shown in FIG. 5.

(2.3.2) Analysis of Bone Cell Differentiation Capacity

Bone cell differentiation assay of the improved umbilical cord-derived stem cells was performed by the following method.

The improved umbilical cord-derived stem cells prepared in (1.1) and (1.3) above were put into osteogenic differentiation media (StemPro® Osteogenesis Differentiation Kit, Life Technology) and the medium was exchanged every three days for two weeks. While culturing. Thereafter, the culture medium was prepared, washed with Ca / Mg free DPBS, and then 4% paraformaldehyde was added and reacted at room temperature for 15 minutes. After the reaction, add purified water, wash, add 1% silver nitrate solution, react for 5 minutes at room temperature, wash with purified water, and add 5% sodium thiosulfate solution at room temperature. The reaction was carried out for 5 minutes. Next, after washing with purified water, 0.1% Nuclear Fast Red Solution was added and reacted at room temperature for 5 minutes. Thereafter, the sample was washed with purified water and analyzed for calcium accumulated samples under a microscope, and the results are shown in FIG. 5.

(2.3.3) Chondrocyte Differentiation Analysis

Cartilage cell differentiation assay of enhanced umbilical cord-derived adherent stem cells was performed by the following method.

Above (1.1) and (1.3) a separate improved cord centrifuged for derived attached stem cells into a 2 x 10 ⁵ dog on a 15 ml tube 1,500 rpm, 5 minutes to remove the supernatant leaving only cell cartilage formation differentiation prepared from Culture medium (Chondrogenesis differentiation media) (StemPro® Chondrogenesis Differentiation Kit, Life Technology) was added and cultured with 3 days intervals for 3 weeks with the cap loosely closed. Cell masses were made into paraffin blocks and then cut into three pieces and subjected to Alcian blue staining. Then, the chondrocytes stained blue with an optical microscope were analyzed, and the results are shown in FIG. 5.

Figure 5 is a view showing the results of analyzing the multipotency of the umbilical cord-derived attached stem cells according to one embodiment.

As shown in FIG. 5, it can be seen that large and small substances (fats), which look like water droplets, are identified as red in the result of adipocyte differentiation assay. In addition, it can be seen from the results of bone cell differentiation that black and white particles calcium lumps are deposited on a pink background. In addition, when the chondrocyte differentiation ability analysis results in differentiation into chondrocytes, glycoproteins indicating the firmness and elasticity of cartilage are secreted, and this part is shown in blue. As a result of the above, the improved umbilical cord-derived attached stem cells according to one embodiment prepared by the method according to (1.1) and (1.3) is differentiated into adipocytes, bone cells, chondrocytes, it can be seen that there is a multipotent ability have.

(2.4) Improved Cord-derived Attached Stem Cells Secretory protein  Analytical Profiling and Quantitative Analysis

In order to analyze the secreted proteins of the improved umbilical cord-derived adherent stem cells prepared in (1.1), a multiplex bead analysis (MILLIPLEX Human Cytokine / Chemokine Panel 1, Merck Millipore, Billerica, Ma, USA) was performed. It was.

Specifically, the culture medium in which the improved umbilical cord-derived stem cells were cultured for 24 hours was incubated with antibody-coated capture beads for 2 hours at room temperature, and washed. The beads are then incubated with biotin-labeled anti-human cytokine and chemokine antibody for 1 hour and streptavidin phycoerythrin for 30 minutes. Incubated. Finally, the secretion protein expression level was analyzed using the Luminex 200 program to wash and quantify the beads, and the results are shown in Table 2.

Category	Cytokine (pg / ml)	3% oxygen partial pressure
Inflammation	IFNr	46
	IL-1a	19
	IL-1b	5
	IL-1ra	30
	IL-2	0
	IL-3	5
	IL-4	8
	IL-5	0
	IL-6	744
	IL-7	20
	IL-8	> 10,000
	IL-9	One
	IL-10	0
	IL-12 (p40)	14
	IL-12 (p70)	2
	IL-13	One
	IL-15	One
	IL-17	0
	TNFa	0
	TNFb	0
	IFNa2	50
	MDC	2
	sCD40L	0
	sIL-2Ra	4
Chemotaxis / Recruitment / Hematopoiesis	Eotaxin	117
	Flt-3 Ligand	6
	G-CSF	3,001
	GM-CSF	46
	MCP-1	> 10,000
	MCP-3	1,033
	MIP-1a	13
	MIP-1b	2
	RANTES	7
	Fractalkine	116
	IP-10	10
Angiogenesis / Tissue remodeling	VEGF	170
Growth factor / Fibrosis	EGF	16
	GRO	> 10,000
	PDGF-AA	0
	PDGF-AB / BB	0
	FGF-2	90
	TGFa	0
Growth factor / Fibrosis	IGF-1 SR	197
Growth factor / Fibrosis	IGFBP3	106.5
Cell adhesion	epCAM	140.5

As shown in Table 2, the improved umbilical cord-derived attached stem cells according to one embodiment is IL-6, IL-8, G-CSF, GM-CSF, MCP-1, MCP-3, VEGF, GRO, IGF It can be seen that the secretion of -1 SR, EpCAM, IGFBP3.

(2.5) Gene Expression Analysis of Enhanced Umbilical Cord-derived Attached Stem Cells

Gene expression of the improved umbilical cord-derived adherent stem cells prepared in (1.1) was analyzed in comparison with bone marrow stem cells.

Specifically, RNA was extracted from the enhanced umbilical cord-derived stem cells and bone marrow stem cells in order to analyze genes specifically expressed in the improved umbilical cord-derived stem cells compared with bone marrow stem cells, followed by labeling and purification. . The labeled cDNA was hybridized to an Illumina Expression BeadChip and the results were obtained. The data are shown in Table 3 and FIG. 6 after statistical processing.

ACCESSIOM	SYMBOL	Bone marrow stem cells / umbilical stem cells.fc	Bone marrow stem cells / umbilical stem cells.
NM_000088.3	COL1A1	-2.181493	15.1373
NM_001552.2	IGFBP4	-2.824265	14.69699
NM_003186.3	TAGLN	-2.531752	14.55202
NM_053056.2	CCND1	3.063696	14.50615
NM_000602.1	SERPINE1	3.478237	14.50551
NM_001080121.1	PRNP	2.289925	14.48807
NM_002966.1	S100A10	-2.008462	14.47747
NM_003900.3	SQSTM1	-2.009802	14.37823
NM_005953.2	MT2A	2.116453	14.37452
NM_001011546.1	DSTN	-2.02492	14.31394
NM_133505.2	DCN	-6.032783	14.27855
NM_006623.2	PHGDH	-2.777842	14.05087
NM_006486.2	FBLN1	-2.856691	14.04878
NM_014220.2	TM4SF1	2.298303	14.04031
NM_003542.3	HIST1H4C	2.048184	14.0358
NM_005928.1	MFGE8	-4.522915	14.01616
NM_000269.2	NME1	2.074511	14.00612
NM_002116.5	HLA-A	-2.348384	13.95215
NM_138440.2	VASN	-2.024294	13.87727
NM_018689.1	KIAA1199	-3.846874	13.85275
NM_003155.2	STC1	-76.224696	10.428439
NM_001031692.1	LRRC17	-48.150192	9.539232
NM_033439.2	IL33	-54.158215	8.92943
NM_000104.2	CYP1B1	349.556757	10.016027

As shown in Table 3 and FIG. 6, COL1A1, IGFBP4, TAGLN, S100A10, SQSTM1, DSTN, DCN, PHGDH, FBLN1, MFGE8, HLA-A, VASN, KIAA1199, STC1, LRRC17, IL33, SNCA, DSG2, NRP2 , PLAT is expressed in enhanced umbilical stem-derived stem cells and CCND1, SERPINE1, PRNP, MT2A, TM4SF1, HIST1H4C, NME1, CXCL6, NTSR1, PTGS2, CYP1B1, TPMT, NAGK, and ANXA4 are found in enhanced umbilical stem-derived stem cells. It can be seen that the expression is lower than the bone marrow stem cells.

Among these genes, COL1A1, an increasing gene, is known to be expressed in collagen of connective tissue including cartilage as alpha-1 type I collagen. However, the gene has not been reported for its relationship with improved umbilical cord-derived adherent stem cells.

Among these genes, IGFBP4, an increase gene, is an insulin-like growth factor binding protein and is known to inhibit various cancer cells. It has been reported that IGFBP4 is detected in the serum of umbilical cord blood, but the gene has not been reported for its association with improved umbilical cord-derived adherent stem cells.

TAGLN, an increasing gene of the genes, is a gene expressed in fibroblasts and smooth muscles, and its function has not been identified yet. Although expression in bone marrow stem cells has been reported, the gene has not been reported for its association with improved umbilical cord-derived adherent stem cells.

Among the genes, CCND1, a decreasing gene, is Cyclin D1. When CCND1 is overexpressed, GND accelerates the cell cycle from G1 to S phase, thereby promoting cell growth. It has been reported to be expressed mainly in cancer cells. Umbilical cord blood stem cells have been reported to inhibit C6 glioma proliferation by decreasing CCND1. However, the gene has not been reported for its association with improved umbilical cord-derived adherent stem cells.

SERPINE1, a reduction gene of the genes, is known as an endothelial plasminogen activator inhibitor and functions as an inhibitor of tissue plasminogen activator (tPA). However, the gene has not been reported for its relationship with improved umbilical cord-derived adherent stem cells.

PRNP, a reduction gene of the genes, is known to be expressed in various tissues as well as the nervous system as a major prion protein (CD230). Abnormalities in the PRNP gene have been reported to cause neurological diseases. However, the gene has not been reported for its relationship with improved umbilical cord-derived adherent stem cells.

In addition, the gene expression of the improved umbilical cord-derived attached stem cells prepared in (1.1) and (1.3) was compared and analyzed in the same manner as above, and the results are shown in Table 4 and FIG. 6.

ACCESSIOM	SYMBOL	Hypoxic Conditions / Oxygen Conditions.fc	Hypoxic conditions / Oxygen conditions.
NM_002966.1	S100A10	2.012102	14.47873
NM_000584.2	IL8	-2.15463	14.32136
NM_000689.3	ALDH1A1	-2.96734	14.21048
NM_004052.2	BNIP3	2.986779	14.13839
NM_000599.2	IGFBP5	2.352086	13.89253
NM_000291.2	PGK1	2.558604	13.8367
NM_000365.4	TPI1	2.104472	13.77506
AV762101		-2.06732	13.66311
NM_133505.2	DCN	2.293184	13.6264
NM_002633.2	PGM1	2.334847	13.19009
NM_000903.2	NQO1	-3.74919	13.18508
NM_004566.2	PFKFB3	2.690257	13.05599
XM_927868.1	LOC644774	3.03134	13.01144
NM_000902.3	MME	2.705127	12.94125
NR_031742.1	MIR1978	2.384465	12.90422
NM_000291.2	PGK1	2.676001	12.84307
NM_006931.1	SLC2A3	2.061179	12.82325
NM_003670.1	BHLHB2	2.329298	12.81616
NM_004331.2	BNIP3L	2.235896	12.7381
NM_000599.2	IGFBP5	3.068411	12.56565
NM_020142.3	NDUFA4L2	10.462756	8.883854

As shown in Table 4 and FIG. 6, S100A10, BNIP3, IGFBP5, PGK1, TPI1, DCN, PGM1, PFKFB3, LOC644774, MME, MIR1978, PGK1, SLC2A3, BHLHB2, BNIP3L, IGFBP5, NDUFA4L and DP3 Highly expressed in enhanced umbilical cord-derived stem cells cultured under hypoxic conditions, and IL8, ALDH1A1, NQO1, DLC1, CTHRC1, and CPA4 were expressed lower in enhanced umbilical cord-derived stem cells cultured under hypoxic conditions than in normal oxygen conditions It can be seen that.

S100A10, an increasing gene, is a S100 calcium-binding protein A10 that regulates cell cycle and differentiation. It is also known to function as exocytosis and endocytosis. Although bone marrow stem cells have been studied as one of the proteins that are highly expressed when differentiated into bone, the gene has not been reported for its association with improved umbilical cord-derived adherent stem cells.

BNIP3, an increase gene of the genes, is known as an increased gene in UCB-MSC when comparing gene expression at the mRNA levels of UCB-MSC (cord blood derived stem cells) and UCB-MNC (cord blood derived blood cells). In addition, after collecting amniotic stem cells under hypoxic conditions, mRNA microarray analysis revealed that the BNIP3 gene is known to be significantly increased in hypoxic conditions, but the gene is reported for its association with improved umbilical cord-derived stem cells. It has never been.

IGFBP5, an increasing gene of the genes, is an insulin-like growth factor binding protein 5, which plays a role in development and is located in the extracellular space. Expression of the IGFBP5 gene in bone marrow stem cells has been reported, but this gene has not been reported for its association with enhanced umbilical cord-derived adherent stem cells.

It is reported that IL8, a decreasing gene of the gene, is secreted from phagocytes and mesenchymal cells when exposed to an inflammatory environment to activate neutrophils that induce chemotaxis. However, the gene has not been reported for its association with improved umbilical cord-derived adherent stem cells.

The reduction gene ALDH1A1 is an aldehyde dehydrogenase 1 family, member A1, which is an enzyme responsible for the major oxidation pathway of alcohol metabolism. The gene has not been reported for its association with enhanced umbilical cord derived stem cells.

3. Anti-inflammatory, Vascular Regeneration and Neuronal Regeneration Effects of Enhanced Umbilical Cord-derived Attached Stem Cell

(3.1) Analysis of anti-inflammatory effects of umbilical cord-derived adherent stem cells

In order to analyze the anti-inflammatory effects of the improved umbilical cord-derived adherent stem cells prepared in (1.1), IL-10 secreted from PBMC proliferation inhibitory activity and activated PBMC was analyzed.

Specifically, PBMC growth inhibition assay was performed as follows. First, the improved umbilical cord-derived adherent stem cells were inoculated in 24 well plates by concentration, and then cultured for 24 hours. Then, PHA was stimulated by adding PHA to CFSE stained PBMC and co-cultured with the improved umbilical cord-derived stem cells for 5 days. Thereafter, according to the presence or absence of the transwell, whether the umbilical cord-derived stem cells secreted by the cytokine secretion is inhibited or whether the inflammation suppressed by direct contact with the cells were distinguished, the results are shown in Figure 7a.

In addition, for IL-10 analysis secreted from the activated PBMC, the conditioned medium of the stomach after collecting the co-culture at 5 hours and 5 days above using a human IL-10 ELISA kit (R & D Systems) IL-10 secretion was measured and the results are shown in Figure 7a.

Figure 7a is a diagram showing the results of analyzing the anti-inflammatory effect of the umbilical cord-derived attached stem cells according to one embodiment.

As shown in Figure 7a, the improved umbilical cord-derived attached stem cells according to one embodiment can be confirmed that the proliferative capacity of PBMC is inhibited compared to the control. In addition, when the umbilical cord-derived attached stem cell: PBMC ratio is 1:10, it can be seen that up to about 30.51 ± 1.74% of the proliferation inhibitory effect is obtained in indirect coculture. In addition, in the IL-10 assay secreted from activated PBMCs, activated PBMCs secrete anti-inflammatory cytokines (IL-10), and enhanced umbilical cord-derived stem cells play a role in PBMCs increasing IL-10 secretion. It can be seen that.

As a result, it can be seen that the improved umbilical cord-derived attached stem cells according to one embodiment can be usefully used for the treatment of inflammatory diseases.

(3.2) Analysis of Blood Vessel Regeneration Effects of Enhanced Umbilical Cord-derived Attached Stem Cells

Vascular endothelial cell proliferation assay was performed to analyze the vascular regeneration effect of the improved umbilical cord-derived attached stem cells prepared in (1.1).

Specifically, a sample was prepared by collecting a culture medium (conditioned medium) of EBM-2 and enhanced umbilical cord-derived attached stem cells. Subsequently, when inoculated with vascular endothelial cells (HUVEC) in a 96 well plate and grown for 1 day, EBM-2 and cultured cells of enhanced umbilical cord-derived adherent stem cells were dispensed and cultured for 4 days. Cyto X ^™ Cell viability assay kit (WST-1) reagent was added in 10% of the medium and reacted in an incubator for 2-3 hours. Then, the proliferation rate of endothelial cells was analyzed by measuring at 450 nm using a microreader, and the results are shown in FIG. 7B.

Figure 7b is a view showing the results of analyzing the blood vessel regeneration effect of the improved umbilical cord-derived attached stem cells according to one embodiment.

As shown in FIG. 7B, when the proliferation ability of the vascular cells cultured in EBM-2 medium is 100%, the vascular cells cultured in the conditioned medium of the improved umbilical cord-derived stem cells proliferate 172 ± 15.22%. You can check it.

As a result of the above can be seen that the improved umbilical cord-derived stem cells according to one embodiment has a blood vessel regeneration effect.

(3.3) Analyzing the neuronal regeneration of umbilical cord-derived stem cells

Neuronal cell proliferation analysis was performed to analyze the neuronal regeneration effect of the improved umbilical cord-derived attached stem cells prepared in (1.1).

Specifically, a sample was prepared by collecting a culture medium (conditioned medium) of MEM and improved umbilical cord-derived attached stem cells. Then, when inoculated neurons (SH-SY5Y) in 96 well plate, and when grown for about one day, the culture medium of MEM and improved umbilical cord-derived attached stem cells were each dispensed and incubated for 4 days. Cyto X ^TM Cell viability assay kit (WST-1) was added in 10% aliquots of the medium and allowed to react in an incubator for 2-3 hours. The proliferation rate of neurons was analyzed by measuring at 450 nm using a microreader, and the results are shown in FIG. 7C.

Figure 7c is a view showing the result of analyzing the neuronal regeneration effect of the improved umbilical cord-derived attached stem cells according to one embodiment.

As shown in FIG. 7C, when the neuronal cell proliferation capacity in the MEM medium is 100%, the neurons cultured in the conditioned medium of the improved umbilical cord-derived attached stem cells proliferate 302 ± 15.97%. .

As a result of the above can be seen that the improved umbilical cord-derived stem cells according to one embodiment has a neuronal regeneration effect.

Claims (22)

1. Enhanced umbilical cord-derived adherent stem cells having one or more properties selected from (a) to (e):

   a) at least one selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17 and IL33 is more expressed compared to bone marrow stem cells;

   b) one or more selected from the group consisting of CCND1, SERPINE1, PRNP and CYP1B1 are less expressed than bone marrow stem cells;

   c) maintaining the shape of adherent fibroblasts during passaging;

   d) ability to differentiate into adipocytes, osteocytes, or chondrocytes; And

   e) selected from the group consisting of CD200 +, Tra-1-60-, CD3-, CD1a-, CD11c-, CD16-, CD86-, CD8a-, CD40-, CD141 +, CD61 +, CD87 +, MIC A / B- and SSEA4 + One or more surface antigen properties.
2. The cell of claim 1, wherein the enhanced umbilical cord-derived adherent stem cells further have one or more properties selected from (f) to (i):

   f) at least one selected from the group consisting of S100A10, BNIP3, IGFBP5, NDUFA4L2, DPYD and SCARA3 is expressed more than in culture under normal oxygen conditions;

   g) at least one selected from the group consisting of IL8, ALDH1A1, DLC1, CTHRC1 and CPA4 is less expressed compared to culture in normal oxygen conditions;

   h) one or more selected from the group consisting of SNCA, DSG2, NRP2, and PLAT is more expressed compared to bone marrow stem cells;

   i) one or more selected from the group consisting of TPMT, NAGK, and ANXA4 is less expressed compared to bone marrow stem cells.
3. The cell of claim 1, wherein the characteristics of a) and b) show a difference in expression level of two or more times compared to bone marrow stem cells by microarray analysis.
4. The cell of claim 2, wherein the characteristics of c) and d) incubated in hypoxic conditions show differences in expression levels of two or more times compared to normal oxygen conditions by microarray analysis and proteomics.
5. The cell of claim 1, wherein the cell has colony forming ability.
6. The method according to claim 1, wherein the cells are IL-6, IL-8, G-CSF, GM-CSF, MCP-3, VEGF, GRO, IFNγ, IL-1a, IL-1b, IL-1ra, IL-3, IL-4, IL-7, IL-9, IL-12 (p40), IL12 (P70), IL-13, IL-14, IFNα2, MDC, sIL-2Ra, Eotaxin, Flt-3 ligand, MCP-1 A cell which secretes proteins of MIP-1a, MIP1b, RANTE, Fractalkine, IP-10, EGF, FGF-2, IGF-1 SR, EpCAM, IGFBP3 or a combination thereof.
7. The cell of claim 1, wherein the cell is derived from Wharton's Jelly tissue of a mammalian umbilical cord.
8. Attaching the separated umbilical cord to a culture vessel and culturing the umbilical cord;

   Separating the improved umbilical cord-derived adherent stem cells by contacting the cultured umbilical cord with a separation enzyme;

   Method for producing an improved umbilical cord-derived attached stem cells comprising the step of passaging the separated enhanced umbilical cord-derived attached stem cells in a medium containing fibroblast growth factor-4 (FGF-4) and heparin.
9. The method of claim 8, wherein the passaging step further comprises treatment of Animal Component Free (ACF) recombinant enzyme prior to cell transplantation for passaging.
10. The method of claim 8, wherein the passaging step is performed under low oxygen conditions compared to 21% of normal oxygen conditions.
11. The method of claim 8, wherein the improved umbilical cord-derived attached stem cells have one or more properties selected from (a) to (e):

    a) at least one selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17 and IL33 is more expressed compared to bone marrow stem cells;

    b) one or more selected from the group consisting of CCND1, SERPINE1, PRNP and CYP1B1 are less expressed than bone marrow stem cells;

    c) maintaining the shape of adherent fibroblasts during passaging;

    d) ability to differentiate into adipocytes, osteocytes, or chondrocytes; And

    e) selected from the group consisting of CD200 +, Tra-1-60-, CD3-, CD1a-, CD11c-, CD16-, CD86-, CD8a-, CD40-, CD141 +, CD61 +, CD87 +, MIC A / B- and SSEA4 + One or more surface antigen properties.
12. The method of claim 8, wherein the passaging is performed up to 3 to 15 passages.
13. The method of claim 8, wherein the isolating enzyme is collagenase.
14. An improved umbilical cord-derived attached stem cell of claim 1, a cell population thereof, or a culture medium thereof, as an active ingredient.
15. Pharmaceutical composition for the treatment or prevention of inflammatory diseases comprising the improved umbilical cord-derived attached stem cells of claim 1, their cell population or culture medium thereof as an active ingredient.
16. The method of claim 15, wherein the inflammatory disease consists of bronchitis, gastritis, arteriosclerosis, arthritis, inflammatory bowel disease (IBD), hepatitis, cholecystitis, fungal infection, gastric ulcer, asthma, atopic dermatitis, tendinitis and nephritis Pharmaceutical composition is selected from the group.
17. Pharmaceutical composition for the treatment or prevention of ischemic disease comprising the improved umbilical cord-derived attached stem cells of claim 1, its cell population or culture medium thereof as an active ingredient.
18. The method of claim 17, wherein the ischemic disease ischemic stroke, myocardial infarction, ischemic heart disease, ischemic brain disease, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, and ischemic leg disease Pharmaceutical composition is selected from the group consisting of.
19. A pharmaceutical composition for treating or preventing neurodegenerative diseases comprising the improved umbilical cord-derived stem cells of claim 1, a cell population thereof, or a culture medium thereof as an active ingredient.
20. The method of claim 19, wherein the neurodegenerative diseases include spinal cord injury, multiple sclerosis, Alzheimer's disease, Frontotemporal dementia, Progressive supranuclear palsy, Corticobasal degeneration, Peak disease (Pick's disease), and boxer dementia (Dementia pugilistica, DP).
21. Use of the improved umbilical cord derived adherent stem cells of claim 1, cell populations thereof or cultures thereof for use in the manufacture of a medicament for use in the treatment or prevention of an inflammatory disease, ischemic disease, or neurodegenerative disease.
22. A method of treating or preventing an inflammatory disease, ischemic disease, or neurodegenerative disease, comprising administering to a subject in need thereof an improved umbilical cord-derived adherent stem cell of claim 1, a cell population thereof, or a culture medium thereof.

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