WO2014046417A1

WO2014046417A1 - Method for preparing mesenchymal stem cell aggregates

Info

Publication number: WO2014046417A1
Application number: PCT/KR2013/008228
Authority: WO
Inventors: Yoon-Sun Yang; Won Il Oh; Hong Bae Jeon; JuEun HA; Soon-Jae Kwon
Original assignee: Medipost Co., Ltd.
Priority date: 2012-09-20
Filing date: 2013-09-11
Publication date: 2014-03-27
Also published as: KR20140038236A

Abstract

Disclosed is a method for preparing mesenchymal stem cell aggregates, which comprises the steps of (1) culturing mesenchymal stem cells in a medium containing calcium in a concentration of from 2.1 to 3.8 mM and magnesium in a concentration of from 1.0 to 3.0 mM under a hypoxic condition with a 2 to 5% of oxygen concentration; and (2) forming aggregates by culturing the mesenchymal stem cells thus obtained in a medium containing serum replacement instead of fetal bovine serum. The mesenchymal stem cell aggregates prepared by the method above have a dense and rigid structure, improved immunological properties and excellent secretion of cytokines, and thus can be used as a cell therapeutic agent safely and effectively.

Description

DESCRIPTION

METHOD FOR PREPARING MESENCHYMAL STEM CELL

AGGREGATES

FIELD OF THE INVENTION

The present invention relates to a method for preparing mesenchymal stem cell aggregates.

BACKGROUND OF THE INVENTION

The term "stem cell" is a generic name for undifferentiated cells obtained from respective tissues of embryos, fetuses and adults, which has the potential of differentiating into a various cell types. Stem cells are characterized by self- renewal, the ability to go through numerous cycles of cell division (while maintaining an undifferentiated state), and potency, the capacity to differentiate into specialized cell types in response to certain stimuli (environment), and even by plasticity, the ability to cross lineage barriers and adopt the expression profile and functional phenotypes of cells that are unique to other tissues.

Stem cells may be classified into pluripotent stem cells, multipotent stem cells and unipotent stem cells according to their potency. Pluripotent stem cells have pluripotency to differentiate into any type of cells. Embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are representative of pluripotent stem cells. Meanwhile, adult stem cells show multipotency and/or unipotency. Among them are hematopoietic stem cells, mesenchymal stem cells, neural stem cells, etc.

In spite of various attempts to utilize human embryonic stem cells having pluripotency as cell therapeutics, the problems of oncogenesis and immune rejection response still remain to be overcome.

Induced pluripotent stem cells (iPS cells) have recently been suggested as a solution to these problems. iPS cells are a type of pluripotent stem cells artificially derived from differentiation-completed adult somatic cells by reprogramming. iPS l cells may avoid the risk of immune rejection response because they are derived entirely from the patient, however, the risk of oncogenesis with iPS cells is still a problem to be solved.

As an alternative, mesenchymal stem cells are being promoted because they exhibit immunomodulatory effects and present no risk of oncogenesis. Mesenchymal stem cells are multipotent stem cells that can differentiate into a variety of cell types, including adipocytes, osteocystes, chondrocytes, myocytes, neurocytes, myocardiocytes, hepatocytes, islet beta cells, vascular cells, etc., and are known to have the function of modulating immune responses.

Mesenchymal stem cells may be isolated from various tissues such as the bone marrow, umbilical cord blood, adipose tissue, etc., but are not sufficiently defined because cell surface markers are somewhat different from one another according to the origin from which the mesenchymal stem cells are derived. On the whole, if they can differentiate into osteoblasts, chondrocytes and myoblasts, have a spindle shaped morphology, and express the surface markers CD73(+), CD105(+), CD34(-) and CD45(-), the stem cells are defined as mesenchymal stem cells. In this context, mesenchymal stem cells of different genetic origins and/or backgrounds do not significantly differ from one another in terms of their definition, i.e., that of a mesenchymal stem cell, but are typically different from each other in terms of in vivo activity. Further, when mesenchymal stem cells are used as exogenous cell therapeutics, a limited pool of mesenchymal stem cells does not allow many choices or available options, even in spite of low in vivo activity.

In addition, the minimum number of mesenchymal stem cells necessary for them to be used as a cell therapeutic in regenerative medicine and/or cell therapy is approximately 1 x 10⁹ cells. In practice, the minimum number is further increased in consideration of experiments for setting proper conditions and determining criteria. The supply of mesenchymal stem cells in such quantities from various origins requires at least ten in vitro passages. In this case, however, the cells become aged and deformed so that they may be unsuitable for use as cell therapeutics.

In this regard, WOl 1/126264 discloses a method for preparing a highly active human mesenchymal stem cell aggregate. WOl 1/126264 demonstrates that mesenchymal stem cell aggregates (also referred to 'mesenchymal stem cell mass') show good in vivo tissue regeneration and treatment efficacy, high in vivo viability, and good differentiation efficiency into tissue cells.

The present inventors have endeavored to study on the mesenchymal stem cell aggregates; and have found that the formation of mesenchymal stem cell aggregates and immunological properties of mesenchymal stem cell aggregates can be enhanced by culturing the mesenchymal stem cells under specific conditions before the cells initiate the formation of aggregates.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide a method for preparing mesenchymal stem cell aggregates, which exhibit improved aggregate formation and immunological properties.

It is another object of the present invention to provide mesenchymal stem cell aggregates prepared by the method, which exhibit improved aggregate formation and immunological properties.

It is a further object of the present invention to provide a cell therapeutic agent comprising the mesenchymal stem cells aggregates.

In accordance with one aspect of the present invention, there is provided a method for preparing mesenchymal stem cell aggregates, which comprises the steps of: (1) culturing mesenchymal stem cells in a medium containing calcium in a concentration of from 2.1 to 3.8 mM and magnesium in a concentration of from 1.0 to 3.0 mM under a hypoxic condition with a 2 to 5% of oxygen concentration; and (2) forming aggregates by culturing the mesenchymal stem cells thus obtained in a medium containing serum replacement instead of fetal bovine serum.

In accordance with another aspect of the present invention, there is provided mesenchymal stem cell aggregates prepared by the method, which exhibit improved aggregate formation and immunological properties.

In accordance with further aspect of the present invention, there is provided a cell therapeutic agent comprising the mesenchymal stem cell aggregates of the present invention.

The method for culturing mesenchymal stem cell aggregates according to the present invention can enhance the formation of mesenchymal stem cell aggregates and immunological properties by culturing the mesenchymal stem cells under specific conditions before the cells initiate the formation of aggregates.

BRIEF DESCRIPTION OF DRAWINGS

FIGs. 1A and IB are graphs showing cell count folds relative to the seeded cell count at 7 days (upper) and cumulative cell counts until 21 days (lower) after umbilical cord blood-derived mesenchymal stem cells derived from two different sources (MSC #1 and #2) were cultured in a-MEM ranging in calcium concentration from 1.8 to 9.3 raM. In the upper graphs, PI to P3 represent numbers of passage.

FIGs. 2A and 2B are graphs showing cell counts after umbilical cord blood- derived mesenchymal stem cells derived from two different sources (MSC #1 and #2) were cultured for 7 days in the presence of a total calcium concentration of from 1.8 to 3.6 mM (upper) and for 6 days in the presence of a total calcium concentration of from 1.8 mM to 4.4 mM (lower).

FIGs. 3A and 3B are graphs showing doubling times when umbilical cord blood-derived mesenchymal stem cells derived from two different sources (MSC #1 and #2) were cultured under various oxygen conditions (normal, 3% and 5%) (upper), and cumulative cell counts until 21 days after the umbilical cord blood-derived mesenchymal stem cells were cultured under the oxygen conditions (lower). In the upper graphs, PI to P3 represent numbers of passage.

FIG. 4 shows doubling times (upper), and cumulative cell counts (lower) after umbilical cord blood-derived mesenchymal stem cells (MSC #1) were cultured in a typical condition (control), in an increased calcium condition (Ca²⁺), in a hypoxic condition, and in a CMH condition. In each graph, P5 to P12 represent numbers of passage, and the CMH condition means a combination of the calcium and magnesium addition condition and the hypoxic condition.

FIG. 5 shows cell viability (upper) and recovery rates (lower) 1 and 2 days after umbilical cord blood-derived mesenchymal stem cells were cultured in a typical condition (control), in a calcium addition condition (Ca ), in a hypoxic condition, and in a CMH condition.

FIGs. 6A and 6B show doubling times (upper), and cumulative cell counts (lower) after umbilical cord blood-derived mesenchymal stem cells (MSC #1 to #4) were cultured in a typical condition (control), and in a CMH condition. In each graph, PI to P9 represent numbers of passage.

FIG. 7 shows mRNA expression levels of the sternness markers Oct4 and nanog and the senescence marker P16 after umbilical cord blood-derived mesenchymal stem cells derived from two different sources (MSC #1 and #2) were cultured in a typical condition (control), in a calcium addition condition (Ca²⁺), in a hypoxic condition, and in a CMH condition.

FIG. 8 shows photographs of umbilical cord blood-derived mesenchymal stem cells stained with SA-p-gal after passages in a typical condition (control) and in a CMH condition (upper), and a graph in which β-gal activity is plotted according to culture conditions after umbilical cord blood-derived mesenchymal stem cells were cultured in a typical condition (control), in a calcium addition condition (Ca²⁺), in a hypoxic condition, and in a CMH condition (lower).

FIG. 9 shows photographs of umbilical cord blood-derived mesenchymal stem cells derived from two different sources (MSC #1 and #2) after the cells cultured in a typical condition (control) and in a CMH condition were induced to differentiate to cartilage and bone.

FIG. 10 shows graphs illustrating whether two different umbilical cord blood- derived mesenchymal stem cells (MSC #1 and #2) cultured in a typical condition (control) and in a CMH condition stimulate responder cells (A), wherein A, B and H represent responder cells, stimulator cells, and PHA, respectively.

FIG. 11 shows graphs of levels of PGE₂ (prostaglandin E₂) released from umbilical cord blood-derived mesenchymal stem cells (MSC #1 and #2) cultured in the conditions of FIG. 10.

FIG. 12 is a graph showing levels of Tsp-2 released from four different umbilical cord blood-derived mesenchymal stem cells (MSC #1 to #4) cultured for 24 hrs in a typical condition (control) and in a CMH condition.

FIG. 13 shows microscopic photographs of aggregates of umbilical cord blood- derived mesenchymal stem cells derived from two different sources (MSC #1 and #2) prepared by cutting the cells in a typical condition and in a CMH condition (3.6 mM Ca²⁺, 1.8 mM Mg²⁺ and 3% 0₂) and forming aggregates by using a hanging drop method described in Example 9. In the figure, IK, 10K and 20K each indicates an aggregate prepared by using l x l O³, l x l O⁴ and 2x 10⁴ cells per 20 μΣ, of medium, respectively.

FIG. 14 shows graphs illustrating immunogenicity of umbilical cord blood- derived mesenchymal stem cell aggregates prepared in accordance with the present invention. Mesenchymal stem cells cultured in a typical condition and in a CMH condition, and mesenchymal stem cell aggregates formed after cultured in a typical condition (I K, 1 OK and 20K) and in a CMH condition (I K, 1 OK and 20K) were used to determine whether or not responder cells (A) were stimulated by these samples. In the figure, A and H refer to responder cells and PHA-L, respectively.

FIG. 15 shows graphs illustrating immunosuppression of umbilical cord blood- derived mesenchymal stem cell aggregates prepared in accordance with the present invention. Mesenchymal stem cells cultured in a typical condition and in a CMH condition, and mesenchymal stem cell aggregates formed after cultured in a typical condition (IK, 10K and 20K) and in a CMH condition (IK, 10K and 20K) were used to determine whether or not these samples show immunosuppression under immune response-induced condition (A+B), where responder cells (A) were allowed to react with stimulating cells (B). In the figure, A, B and H refer to responder cells, stimulating cells and PHA-L, respectively.

FIG. 16 shows graphs illustrating cytokine secretion of umbilical cord blood- derived mesenchymal stem cell aggregates, which demonstrate the amounts of Tsp-2 and VEGF secreted from mesenchymal stem cells cultured in a typical condition and in a CMH condition, and mesenchymal stem cell aggregates formed after cultured in a typical condition (I K, 10K and 20K) and in a CMH condition (I K, 10K and 20K).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with a preferred embodiment, the present invention provides a method for preparing mesenchymal stem cell aggregates, which comprises the steps of: (1 ) culturing mesenchymal stem cells in a medium containing calcium in a concentration of from 2.1 to 3.8 mM and magnesium in a concentration of from 1.0 to 3.0 mM under a hypoxic condition with a 2 to 5% of oxygen concentration; and (2) forming aggregates by culturing the mesenchymal stem cells thus obtained in a medium containing serum replacement instead of fetal bovine serum.

As used herein, the term "aggregate" refers to a spherical stem cell aggregation formed by culturing stem cells, and used interchangeably with "stem cell mass" or "spheroid" in the art.

The culturing method of the present invention may be applied to mesenchymal stem cells of various origins. Examples of the mesenchymal stem cells useful in the present invention include those derived from umbilical cord blood, bone marrow, lipid, muscle, skin, amniotic fluid, umbilical cord, or teeth, but are not limited thereto. In one preferred embodiment of the present invention, the culturing method of the present invention is applied to umbilical cord blood-derived mesenchymal stem cells.

In addition, the mesenchymal stem cells to which the culturing method of the present invention can be applied may be derived from various subjects. For example, the mesenchymal stem cells useful in the present invention may be obtained from mammals including humans, but are not limited thereto. In one preferred embodiment of the present invention, mesenchymal stem cells of human origin are used.

In the method for preparing mesenchymal stem cell aggregates in accordance with the present invention, Step (1) is characterized by culturing mesenchymal stem cells in a medium containing calcium in a concentration of from 2.1 to 3.8 mM and magnesium in a concentration of from 1.0 to 3.0 mM under a hypoxic condition with a 2 to 5% of oxygen concentration. The culture medium may be prepared from a typical culture medium for stem cells by adjusting the concentrations of calcium and magnesium. Examples of the typical culture medium include Dulbecco's modified eagle medium (DMEM), minimal essential medium (MEM), a-MEM, McCoys 5A medium, eagle's basal medium, CMRL (Connaught Medical Research Laboratory) medium, Glasgow minimal essential medium, Ham's F-12 medium, IMDM (Iscove's modified Dulbecco's medium), Leibovitz's L-15 medium, RPMI (Roswell Park Memorial Institute) 1640 medium, medium 199, and Hank's medium 199, but are not limited thereto.

Optionally, the culture medium may or may not contain serum. In addition, a serum replacement may be used, instead of serum, in the culture medium.

In one embodiment of the present invention, the culture medium contains 5 to 30% of fetal bovine serum (FBS). In another embodiment, the culture medium contains a serum replacement. In addition to a commercially available product, various growth factors in a human serum or a human platelet lysate, including PDGF, TGF, IGF, and cytokines of a family of such proteins may be used as the serum replacement.

In the culturing method of the present invention, calcium functions to promote the proliferation of mesenchymal stem cells, with the suppression of immunogenicity and the stimulation of cytokine secretion. In this regard, calcium may be used in a concentration of from 2.1 to 3.8 mM in the medium, preferably in a concentration of from 3.3 to 3.8 mM, and more preferably in a concentration of approximately 3.6 mM. For instance, when a-MEM is adopted as the culture medium, calcium may be added in a concentration of from 0.3 to 2.0 mM, preferably in a concentration of from 1.5 to 2.0 mM, and more preferably in a concentration of approximately 1.8 mM because the medium already contains 1.8 mM of calcium. Likewise, the calcium concentration to be added to achieve the desired concentration necessary for implementing the culturing method of the present invention can be readily calculated in consideration of the calcium concentration of a medium itself, taken from among typical media.

In the culture medium of the present invention, magnesium is employed to prevent the precipitation of calcium. Magnesium may be used in a concentration of from 1.0 to 3.0 mM in the medium, and preferably in a concentration of approximately 1.8 mM. For example, when magnesium is present in a concentration of less than 1.0 mM in the culture medium, calcium is apt to precipitate. On the other hand, a magnesium concentration higher than 3.0 mM in the culture medium is likely to block the formation of the extracellular matrix (ECM), interfere with the adherence of the cells to the bottom of the culture dish, thus rendering them susceptible to shear stress, and increase intracellular mineralization. For instance, when a-MEM is adopted as the culture medium, magnesium may be added in a concentration of from 0.2 to 2.2 mM, and preferably in a concentration of 1.0 mM because the medium already contains 0.8 mM of magnesium. Likewise, the magnesium concentration to be added to achieve the desired concentration necessary for implementing the culturing method of the present invention can be readily calculated in consideration of the magnesium concentration of a medium itself, taken from among typical media.

Thus, the culture medium according to a preferred embodiment of the present invention may be based on a-MEM supplemented with 5 to 30% of fetal bovine serum (FBS), 0.3 to 2.0 mM of calcium, and 0.2 to 2.2 mM of magnesium, thus calcium and magnesium amounting to a total of from 2.1 to 3.8 mM, and from 1.0 to 3.0 mM, respectively.

The hypoxic condition of Step (1) in accordance with the present invention, as compared to a normoxic condition, efficiently promotes the proliferation of mesenchymal stem cells, with the suppression of immunogenicity and the stimulation of cytokine secretion. In this context, the hypoxic condition is an atmosphere with an oxygen content of from 2 to 5%. A problem with an oxygen concentration below 2% or over 5% is a significant decrease in the proliferation of mesenchymal stem cells. In one preferred embodiment of the present invention, mesenchymal stem cells are cultured in an atmosphere of approximately 3% of oxygen. The hypoxic condition may be achieved by adjusting the oxygen concentration of a cell incubator. For example, an incubator may be purged with nitrogen (100%) or nitrogen/carbon dioxide (95%/5%) to adjust the normoxic atmosphere into a hypoxic atmosphere. The oxygen concentration in an incubator may be monitored by an oxygen sensor installed on the incubator.

Except for the aforementioned conditions of the present invention, mesenchymal stem cells may be cultured in a conventional manner. For example, mesenchymal stem cells may be cultured in a three-dimensional bioreactor or spinner or a typical adherent culture vessel.

When the primary feature for the concentration of calcium and magnesium is combined with the secondary feature for the hypoxic condition in Step (1) of the present invention, a synergistic effect can be obtained. That is, a combination of the concentration of calcium and magnesium and the hypoxic condition allows mesenchymal stem cells to proliferate more efficiently, with a higher improvement in the suppression of immunogenicity and the stimulation of cytokine secretion, compared to the individual conditions. For example, under the combined conditions, mesenchymal stem cells proliferate 1.5- to 5-fold further, with a 1- to 3- fold decrease in immunogenicity, and a 1.5- to 3-fold increase in cytokine secretion, compared to individual conditions. The combined condition for the culturing method of the present invention is referred to as "CMH condition" (calcium + magnesium + hypoxic condition).

In the method for preparing mesenchymal stem cell aggregates in accordance with the present invention, Step (2) is characterized by forming aggregates by culturing mesenchymal stem cells obtained in Step (1) in a medium containing serum replacement instead of fetal bovine serum.

Examples of culture medium which may be used for forming aggregates in Step (2) include Dulbecco's modified eagle medium (DMEM), minimal essential medium (MEM), a-MEM, McCoys 5A medium, eagle's basal medium, CMRL (Connaught Medical Research Laboratory) medium, Glasgow minimal essential medium, Ham's F-12 medium, IMDM (Iscove's modified Dulbecco's medium), Leibovitz's L-15 medium, RPMI (Roswell Park Memorial Institute) 1640 medium, Medium 199, and Hank's medium 199, but are not limited thereto.

In one embodiment of the present invention, the culture medium free of fetal bovine serum (FBS) does not include basic fibroblast growth factor (bFGF) but may additionally contain a serum replacement. The serum replacement may comprise serum albumin, transferrins, hemoglobin beta chains, etc. as a main ingredient. Specifically, the serum replacement may include serum albumin precursor, apolipoprotein A-I precursor, transthyretin precursor, serotransferrin precursor, antithrombin III, plasma retinol-binding protein (PRBP), serotransferrin, vitamin D- binding protein precursor, hemoglobin beta chain, anionic trypsin II precursor, alpha- 1 -antiproteinase, alpha-2-HS-glycoprotein precursor, etc. Any conventional serum replacement known in the art may be employed in the present invention, e.g., KnockOut™ SR (Invitrogen). In addition to a commercially available product as the serum replacement, various growth factors in a human serum or a human platelet lysate, including PDGF, TGF, IGF, and cytokines may be used.

In the present invention, the culture of the human mesenchymal stem cells to form the spherical cell aggregate may be carried out in a culture drop placed against gravity. The spherical cell aggregate may be formed to include 300 to 30,000 cells per culture drop (approximately 20μί), preferably 1 ,000 to 30,000 cells per culture drop, so as to obtain a spherical cell aggregate having a high therapeutic efficacy.

The culturing method of stem cells against gravity results in obtaining a number of stem cell aggregates having a uniform size, which enhance the therapeutic effectiveness.

Further, the culture of the mesenchymal stem cells to form the aggregate may be carried out by suspension culture employing a low-attachment culture dish. Also, the aggregate may be formed by employing a three-dimensional bioreactor.

In a preferred embodiment of the present invention, the mesenchymal stem cells may be cultured at room temperature, for example, 30 to 40 °C, preferably about 37 °C, for 12 to 72 hours under atmospheric condition with 5% C0₂.

After the formation of the spherical cell aggregates, the produced aggregates may be separated by using a strainer.

Further, the present invention provides mesenchymal stem cell aggregates prepared by the method. The mesenchymal stem cell aggregates in accordance with the present invention have a dense and rigid structure, implying that aggregates can be easily formed owing to the increased expression of adhesive molecules present or inducible on the surface of the mesenchymal stem cells. In addition, the mesenchymal stem cell aggregates in accordance with the present invention have reduced immunogenicity, improved immunosuppression and good secretion ability of cytokines (e.g., Tsp-2 and VEGF), and thus, may be used safely and effectively as a cell therapeutic agent.

Further, the present invention provides a cell therapeutic agent comprising the mesenchymal stem cell aggregates. The cell therapeutic agent of the present invention finds applications in the regeneration or protection of adipocytes, osteocytes, chondrocytes, myocytes, neurocytes, cardiomyocytes, hepatocytes, islet beta cells, vascular cells, or pneumocytes. In addition, the cell therapeutic agent of the present invention is useful for one selected from the group consisting of the treatment of pulmonary diseases; the suppression or treatment of lung disease- induced inflammation; the regeneration of pulmonary tissues; and the suppression of pulmonary fibrosis. Particularly, it can be used to suppress or improve pulmonary disease-induced inflammation and fibrosis. Further, the cell therapeutic agent of the present invention can be applied to the therapy of cardiovascular diseases or the regeneration of cartilage. Moreover, the cell therapeutic agent of the present invention can reduce immune responses, immune cell penetration, or immunogenicity; improve immunomodulative functions; and suppress inflammatory reactions. Also, the cell therapeutic agent of the present invention is applied to therapy of autoimmune diseases, or graft- vs-host diseases.

Still further, the present invention provides a use of the cell a cell therapeutic agent comprising the mesenchymal stem cell aggregates in the manufacture of a medicament for: (1) regeneration or protection of adipocytes, osteocytes, chondrocytes, myocytes, neurocytes, cardiomyocytes, hepatocytes, islet beta cells, vascular cells, or pneumocytes; (2) one selected from the group consisting of treatment of pulmonary diseases, suppression or treatment of lung disease-induced inflammation, regeneration of pulmonary tissues, and suppression of pulmonary fibrosis; (3) regeneration of cartilage; or (4) therapy of autoimmune diseases, or graft-vs-host diseases.

Hereinafter, the present invention is described in more detail. The following Examples are given for the purpose of illustration only, and are not intended to limit the scope of the invention.

For use in the present invention, human cord blood-derived mesenchymal stem cells were obtained from Medipost Co. Ltd., Korea. The cells may be prepared by collecting umbilical cord blood, isolating mesenchymal stem cells from umbilical cord blood, and culturing the mesenchymal stem cells, as illustrated below.

Umbilical cord blood may be collected from the umbilical vein which is expelled out of the uterus either while the placenta remains within the uterus after normal spontaneous vaginal delivery or once the placenta has been expelled from the uterus after cesarean section.

After neonatal birth, the umbilical vein which is expelled from the uterus and by which the newborn is connected to the placenta must be aseptically treated before collecting umbilical cord blood therefrom.

Umbilical cord blood is withdrawn from the umbilical vein into a bag containing an anticoagulant through a syringe.

Methods of isolating mesenchymal stem cells from umbilical blood and culturing the cells are disclosed in Korean Patent No. 10-0494265, and many reports (Pittinger MF, Mackay AM, et al., Science, 284: 143-7, 1999; Lazarus HM, Haynesworth SE, et al., Bone Marrow Transplant, 16: 557-64, 1995). One of them is briefly described below.

Monocytes are separated by centrifuging the collected umbilical cord blood and washed several times to remove impurities therefrom. Then, the monocytes are seeded at a proper density into a culture vessel and allowed to grow with the formation of a single layer. Mesenchymal stem cells are morphologically homogeneous and grow while forming colonies comprising spindle-shaped cells, as observed under a phase-contrast microscope. Then, the cells are cultured with passage upon confluence until a necessary number of cells are obtained.

EXAMPLE 1: Proliferative Capacity of Umbilical Cord Blood-Derived Mesenchymal Stem Cells According to Calcium Concentration

To examine the proliferative capacity thereof according to calcium concentration, umbilical cord blood-derived mesenchymal stem cells were cultured in the presence of various concentrations of calcium.

Umbilical cord blood-derived mesenchymal stem cells (MSC #1 and #2) which had been collected after delivery with the informed consent of different mothers and stored in a frozen state were thawed, and cultured at 37 °C in a-MEM (Invitrogen, USA) supplemented with 10% FBS under a 5% C0₂ condition in an incubator (hypoxic/C0₂ incubator, Thermo Scientific #3131). When the cells were grown to 80 to 90% confluency, they were separated into single cells by treatment with trypsin. To a-MEM (supplemented with 10% FBS; containing 1.8 raM calcium and 0.8 mM magnesium), various concentrations (0 mM, 1.5 mM, 3 mM, 4.5 mM, 6 mM, and 7.5 mM) of calcium were added so that the calcium concentrations of the medium was adjusted into: 1.8 mM, 3.3 mM, 4.8 mM, 6.3 mM, 7.8 mM, and 9.3 mM. The mesenchymal stem cells were inoculated at a density of 5,000 cells/cm into the media. In order to prevent calcium-induced precipitation, magnesium was added in a concentration of 1 mM to each medium (containing a total magnesium concentration of 1.8 mM). The cells were cultured in a 21% (v/v) oxygen (normoxia) condition, with passages upon 80 to 90% confluency. They were counted every passage, using a Cellometer Auto T4 cell counter (Nexelcom, Lawrence, MA, USA). The results are given in FIGs. 1A and IB. FIGs. 1A and IB are graphs showing cell count folds relative to the seeded cell count at 7 days (upper) and cumulative cell counts until 21 days (lower) after umbilical cord blood- derived mesenchymal stem cells derived from two different sources (MSC #1 and #2) were cultured in ct-MEM to which calcium was further added in various concentrations of from 0 to 7.5 mM.

As can be seen in FIGs. 1A and IB, the proliferative capacity of the cells peaked when calcium was further added in a concentration of 1.5 mM (a total calcium concentration of 3.3 mM), which was also observed in the same pattern over passages. Upon the addition of 3 mM or higher calcium (a total calcium concentration of 4.8 mM or higher in media), the proliferative capacity was gradually decreased.

In order to determine an optimal calcium concentration, calcium was added in further fractioned concentrations to the maximum of 3 mM. The results are shown in FIGs. 2 A and 2B. FIGs. 2 A and 2B are graphs showing cell counts after umbilical cord blood-derived mesenchymal stem cells derived from two different sources (MSC #1 and #2) were cultured for 7 days in the presence of a total calcium concentration of 1.8 mM, 2.1 mM, 2.4 mM, 2.7 mM, 3.0 mM, 3.3 mM and 3.6 mM (upper), and for 6 days in the presence of a total calcium concentration of 1.8 mM, 3.4 mM, 3.6 mM, 3.8 mM, 4.0 mM, 4.2 mM, and 4.4 mM (lower).

As can be seen in the graphs, the proliferative capacity increased over an added calcium concentration range from 0 to 1.8 mM (total concentrations of from 1.8 to 3.6 mM in media), and then started to decrease when the added calcium concentration exceeded 1.8 mM (a total calcium concentration of 3.6 mM in media). From these results, it is understood that the optimal calcium concentration for allowing the maximal proliferation of mesenchymal stem cells is 3.6 mM in a medium. Thus, it is advantageous in terms of proliferative capacity that mesenchymal stem cells are cultured in a typical medium containing calcium preferably in a concentration of from 2.1 to 4.3 mM, and more preferably in a concentration of from 3.3 to 3.8 mM.

EXAMPLE 2: Proliferative Capacity of Umbilical Cord Blood-Derived Mesenchymal Stem Cells According to Oxygen Concentration

To examine the proliferative capacity thereof according to oxygen concentration, umbilical cord blood-derived mesenchymal stem cells were cultured in the presence of various concentrations of oxygen.

Specifically, umbilical cord blood-derived mesenchymal stem cells were cultured in the same manner as in Example 1 under 3% or 5% of oxygen, or under a normoxic (oxygen level 21% in air) condition, with the exception that neither calcium nor magnesium was further added to a 10% FBS-supplemented a-MEM. The results are given in FIGs. 3A and 3B. FIGs. 3A and 3B are graphs showing times it took for the cells to double in number when umbilical cord blood-derived mesenchymal stem cells derived from two different sources (MSC #1 and #2) were cultured under various oxygen conditions (normal, 3% and 5%) after 1, 2 and 3 rounds of passage (upper), and cumulative cell counts until 21 days after the umbilical cord blood-derived mesenchymal stem cells were cultured under the oxygen conditions (lower).

As can be seen in these graphs, the proliferative capacity was measured to be higher under the hypoxic conditions than the normoxic conditions, although there were differences between batches. Particularly, the proliferative capacity peaked at an oxygen level of 3%, which was observed in the same pattern for the cells which had been cultured with many rounds of passage. In addition, the cells were examined for proliferative capacity under further fractioned oxygen conditions to a maximum of 5%. An oxygen level of from 2 to 5% was preferred (data not shown).

EXAMPLE 3: Proliferative Capacity of Umbilical Cord Blood-Derived Mesenchymal Stem Cells According to Combination of Calcium (inclusive of Magnesium) and Oxygen Conditions

An examination was made of the proliferative capacity of umbilical cord blood-derived mesenchymal stem cells according to combinations of calcium (inclusive of magnesium) and oxygen concentration conditions. The cells were cultured in a typical condition (control), in the presence of externally added calcium (inclusive of magnesium), in a hypoxic condition, and in an externally added calcium (inclusive of magnesium)/hypoxic condition (hereinafter referred to as "CMH"). In this regard, the media contained calcium and magnesium at total concentrations of 3.6 and 1.8 mM, respectively (1.8 mM calcium and 1 mM magnesium additionally added). The hypoxic condition was set forth at an oxygen level of 3%. The cells were cultured in a manner similar to that of Example 1. After 5 passages (P5) in the typical condition, the mesenchymal stem cells were cultured with 7 rounds of passages (PI 2) in the CMH condition at regular intervals of 7 days between passages.

The results are given in FIG. 4. FIG. 4 shows doubling times (day) of the cells (upper), and cumulative cell counts (lower) after passages under the conditions.

As is understood from the data of FIG. 4, the proliferative capacity of the cells was significantly increased when they were cultured in the CMH condition, compared to the hypoxic condition or the calcium addition condition. This effect was observed in the same pattern over many rounds of passage. Experiments with various batches of cells showed similar results although there were differences to some degree. Thus, these results demonstrate that the CMH condition of the present invention is very effective for proliferating umbilical cord blood-derived mesenchymal stem cells.

EXAMPLE 4: Viability and Recovery Rate of Umbilical Cord Blood- Derived Mesenchymal Stem Cells According to Culture Condition

An examination was made of the effect of the CMH condition of the present invention on the viability and recovery rate of umbilical cord blood-derived mesenchymal stem cells. For this, umbilical cord blood-derived mesenchymal stem cells (MSC #1) were cultured in a typical condition (control), in a hypoxic condition (3%), in an increased calcium condition (1.8 mM; a total calcium level of 3.6 mM in a medium), and in a CMH condition (3% 0₂ + 1.8 mM calcium added + 1 mM magnesium added), detached from culture vessels, and washed three times with and suspended in a fundamental medium (cc-MEM). While being maintained at room temperature, the cell suspensions were examined for viability and recovery rate with time. Cell viability was expressed as a percentage of live cells to dead cells after the cells collected and suspended in a fundamental medium were stained with trypan blue and total cells including live cells stained blue in a predetermined volume (10 to 20 μί,) of the suspension were counted using a hemocytometer. The recovery rate was expressed as a percentage of live cell counts post-culture to pre-culture.

The results are given in FIG. 5. FIG. 5 shows cell viability (upper) and recovery rates (lower) one and two days after umbilical cord blood-derived mesenchymal stem cells were cultured in the conditions.

As can be seen in FIG. 5, the cells were observed to exhibit higher viability and recovery rate when they were cultured in the hypoxic condition or the increased calcium condition than in the typical condition, and even higher viability and recovery rate when they were cultured in the CMH condition. The same results were obtained with umbilical cord blood-derived mesenchymal stem cells derived from different sources although there were some differences therebetween to some degree. These data, taken together, indicate that the CMH condition is advantageous over a typical condition, or individual conditions, in increasing the viability of umbilical cord blood-derived mesenchymal stem cells to recover a greater number of cells.

Mesenchymal stem cells (MSC #1 to #4) were cultured with passage in a typical condition and in the CMH condition, and examined for proliferative capacity. The results are given in FIGs. 6A and 6B which show doubling time (upper) and cumulative cell counts (lower).

As can be seen in the graphs, the CMH condition significantly reduced the doubling time, an index for cell proliferation, over many rounds of passage, compared to the control. In addition, as is apparent from the data of the cumulative growth curves, a much greater number of mesenchymal stem cells, even though derived from the same source, were obtained in the CMH condition. The same results were obtained from experiments with different umbilical cord blood-derived mesenchymal stem cells although there was a difference therebetween to some degree. These data indicate that the CMH condition induces mesenchymal stem cells to proliferate with better efficiency. Particularly, an even greater number of mesenchymal stem cells were produced when the CMH condition was applied to an initial passage of umbilical cord blood-derived mesenchymal stem cells.

EXAMPLE 5: Assay for Stemness and Senescence of Umbilical Cord Blood-Derived Mesenchymal Stem Cells According to Culture Condition To examine why the CMH condition improves the proliferation of umbilical cord blood-derived mesenchymal stem cells, their stemness and senescence, which are associated with the proliferation of stem cells, were assayed.

For this, umbilical cord blood-derived mesenchymal stem cells were cultured in a typical condition and in a CMH condition, as in Example 3. The cells were detached with trypsin when they reached 80 to 90% confluency. After removal of the media by centrifugation, the cells were washed with PBS and recovered by centrifugation. This procedure was repeated twice to completely remove media from the cells. Subsequently, RNA was isolated using an RNA isolation kit (Invitrogen) according to the protocol of the manufacturer. The RNA was reverse transcribed into cDNA in the presence of the reverse transcriptase SuperScript™III (Invitrogen). Real-time PCR was carried out on the cDNA using primers specific for the stemness markers Oct4 and nanog, the senescence marker PI 6, and GADPH. The PCR started with denaturation at 95 °C for 10 min, and was performed with 30 cycles of 95 °C for 10 sec, 62 °C for 30 sec, and 72 °C for 10 sec in a LightCycler 480 Real-Time PCR System instrument (Roche).

[TABLE 1]

Primers for RT-PCR

F; AGCCACCATCGCTCAGACAC (SEQ ID NO: 7)

|R; GCCCAATACGACCAAATCC (SEQ ID NO: 8)

The levels of RNA obtained by the RT-PCR were normalized to that of GAPDH before the expression levels of RNA for each marker in the cells cultured in the typical condition and the CMH condition were compared (relative analysis, ddCT method). -

The results are given in FIG. 7. FIG. 7 shows mRNA expression levels of two different umbilical cord blood-derived mesenchymal stem cells (MSC #1 and #2).

As can be seen in FIG. 7, the expression levels of the sternness markers Oct4 and nanog were higher in the umbilical cord blood-derived mesenchymal stem cells cultured in the CMH condition than in a typical condition (control) and than in individual conditions. The senescence marker PI 6 showed an inverse expression pattern to that of Oct4. These results indicate that the CMH condition maintains the sternness of mesenchymal stem cells while suppressing the senescence, thus improving proliferative capacity.

To confirm the ability of the CMH condition to suppress the senescence of mesenchymal stem cells, the following experiments were carried out. Umbilical cord blood-derived mesenchymal stem cells were cultured in a typical condition and in a CMH condition as in Example 3, with 7 to 8 passages. After removal of the media, the cells were washed once with PBS, and incubated at room temperature for 3 to 5 min with 1 mL of a lx fixation solution (Cell Signaling Technology). The fixation solution was removed from the cells which were then washed twice with 2 mL of PBS. Subsequently, the cells were incubated for 2 to 24 hrs with 1 mL of a dye solution for β-galactosidase (Cell Signaling Technology) in a 37 °C incubator. After removal of the dye solution therefrom, the cells were washed with 1 mL of PBS, and the resulting stained senescent cells were counted under the inverted microscope ECLIPSE TE2000-U (Nikon Co., Kanagawa, Japan).

The results are given in FIG. 8. FIG. 8 shows microphotographs of cells after staining with SA-P-gal (upper), and graphs of SA-P-gal activity (lower). The SA-P-gal activity was determined by calculating the ratio of stained cells to total cells counted on a photograph taken at 40- to 100-fold magnification. As is apparent from FIG. 8, the progression of senescence in the mesenchymal stem cells was retarded further in the CMH condition than in the calcium addition condition or the hypoxic condition, and much further than in the typical condition.

Taken together, the data obtained above demonstrate that the CMH condition of the present invention maintains sternness and suppresses senescence more efficiently than do the typical conditions or the individual conditions, whereby the mesenchymal stem cells can proliferate with high efficiency.

EXAMPLE 6: Differentiation Potential and Marker Expression of Umbilical Cord Blood-Derived Mesenchymal Stem Cells According to Culture Condition

An examination was made of the effect of the CMH condition on the property of umbilical cord blood-derived mesenchymal stem cells. To this end, mesenchymal stem cells were assayed for differentiation potential and marker expression by chondrogenic induction and osteogenic induction.

Umbilical cord blood-derived mesenchymal stem cells obtained from two different sources (MSC #1 and #2) were cultured in a typical condition (control) and in a CMH condition, as in Example 3, before they were induced to differentiate into cartilage and bone, as follows. Then, differentiation into cartilage and bone was evaluated using a staining method.

Chondrogenic induction

For use in chondrogenic induction, cells were placed in a population of 2 to 2.5x 10^s cells in a 15 mL conical tube, and centrifuged to give a cell pellet. It was washed with D-PBS and suspended in 200 to 250 μΐ, of a differentiation medium [high glucose DMEM (Gibco, cat#. 11995), 10 ng/mL TGFp-3 (Sigma, cat#. T5425, 2 μξ), 500 ng/mL BMP-6 (R&D, cat#. 507-BP, 20 μg), 50 μg/mL ascorbic acid (Sigma, cat#. A8960), 50 mg/mL (1 : 100) ITS™+ Premix (BD, cat#. 354352), 40 μg/mL L-proline (Sigma, cat#. P5607), 100 μg/mL sodium pyruvic acid (Sigma, cat#. P8574), 100 nM dexamethasone (Sigma, cat#. D2915)], and the cell suspension was aliquoted into tubes. These tubes were centrifuged at 1,500 rpm for 5 min, after which the cells were cultured for 4 weeks in a C0₂ incubator at 37 °C, with the tubes opened slightly, to induce differentiation into cartilage. The differentiation medium was substituted by half with a fresh one, twice a week.

Cartilage staining protocol

After the chondrogenic induction, the cells were centrifuged, washed with PBS, and fixed at room temperature for 0.5 to 1 hr in 4% paraformaldehyde. Subsequently, the cells were washed two or three times with distilled water, and prepared into sections (4 to 5 μηι thick) using a cryosection method. The sections were immersed for 3 to 5 min in 95% ethanol, and washed twice with water. After being stained for 7 min with 0.1% safranin O, the cells were washed twice with 70% ethanol, once with 70% ethanol, twice with 95% ethanol, once with 95% ethanol, and twice with 100% ethanol, immersed for 3 min in a xylene substrate solution, and dried. Thereafter, the stained cells were covered with a lipid-soluble mounting solution and observed. The chondrogenic induction was evaluated by comparing the color (violet), the size of differentiated pellets, and the lacuna structure formed.

Osteogenic induction

For use in osteogenic induction, the cells were plated at a density 500 to 1000 cells/well into 6-well plates, and 2 to 4 days later, the medium was substituted with an osteogenic induction medium (β-glycerol phosphate 2.1604 g, L-ascorbic acid-2- phosphate 0.012805 g, dexamethasoneAJVAB 0.6 mg, gentamycin (10 mg mL) 5 mL and FBS 100 mL in 1 L of a-MEM). The cells were cultured for 2 to 3 weeks with the differentiation medium substituted with a fresh one every three days. The chondrogenic induction was evaluated by an ALP staining method.

Bone staining protocol

The differentiated cells were washed twice with PBS and incubated for 30 to 45 sec in a fixation solution (40% acetone). They were washed again two or three times with distilled water and incubated for 30 min with an alkaline staining solution (Fast violet B salt) in a dark place. Then, the cells were washed twice with distilled water, and treated for 10 to 20 sec with Mayer's hematoxylin solution. After removal of the staining solution therefrom, the cells were washed with tap water, dried, covered with a lipid-soluble mounting solution, and observed. Because osteoblasts are stained dark brown due to the activation of intracellular alkaline phosphatase, the chondrogenic induction was evaluated by the degree of staining.

The results are given in FIGs. 9A and 9B. As can be seen in FIGs. 9A and 9B, there were no significant differences in chondrogenic induction and osteogenic induction between the mesenchymal stem cells cultured in the typical condition and in the CMH condition.

Meanwhile, immunophenotypes of the cell surface antigens on the umbilical cord blood-derived mesenchymal stem cells cultured according to the method of the present invention were examined. In this context, the expression of the surface markers (CD34, CD73, CD45, and CD 105) was analyzed using FACS.

Umbilical cord blood-derived mesenchymal stem cells cultured in a typical condition and in the CMH condition were trypsinized, and washed three times with PBS containing 2% FBS. They were reacted with the hematopoietic cell-associated antigens CD34 and CD45, both conjugated with FITC (fluorescein isothiocyanate), the immunomodulation-associated antigen CD73 conjugated with PE (phycoerythrin), and the angiogenesis-associated antigen CD 105 conjugated with PE. Afterwards, the cells were additionally marked with a secondary antibody (IgG-FITC; Jackson ImmunoResearch, West Grove, PA, USA) in a manner similar to Western blotting, followed by detecting the signal of the secondary antibody using FACS to ratios of the cells expressing the markers to total cells. After the reaction, the signals were analyzed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA), and the software CELLQUEST.

The results are summarized in Table 2, below.

[TABLE 2]

As is understood from the data of Table 2, there were no significant differences in the expression of marker proteins between cells cultured in the CMH condition and in the typical condition.

Taken together, the data obtained above demonstrate that the CMH condition of the present invention has no significant influence on the fundamental properties of umbilical cord blood-derived mesenchymal stem cells.

EXAMPLE 7: Comparison of Immunogenicity and Immunosuppression of Umbilical Cord Blood-Derived Mesenchymal Stem Cells According to Culture Condition

Immunological properties of umbilical cord blood-derived mesenchymal stem cells according to culture conditions were evaluated using a mixed lymphocyte reaction (MLR) as follows.

For a negative control, umbilical cord blood-derived mesenchymal stem cells cultured in the presence of 10 μg/mL mitomycin C (Sigma- Aldrich, St Louis, MO, USA) in a typical condition and in a CMH condition were separately seeded at a density of 2>< 10⁴ cells/well into 96-well plates, responder cells (peripheral blood monocytes (expressed as "A"); ALLCELLS, Emeryville, CA) at a density of 1 >< 10⁵ cells/well, and stimulator cells (unrelated peripheral blood monocytes (expressed as "B"); ALLCELLS, Emeryville, CA) at a density of 1 * 10⁵ cells/well. As a positive control (1), peripheral blood monocytes treated with 10 μg/mL PHA-L (expressed as "H"; Roche Diagnostics GmbH, Mannheim, Germany) were added at a density of l x lO⁵ cells/well to 96-well plates. For a positive control (2), each of the responder cells and the stimulator cells were added at a density of 1 ^χ 10⁵ cells/well. In a test group, mesenchymal stem cells were incubated with peripheral blood monocytes, PHA-L-stimulated peripheral blood monocytes, or a combination of the responder cells and the stimulator cells, each monocyte being used at a density of 1 x 10⁵ cells, for 5 days, and the proliferation and colony formation of the responder cells were observed under a microscope. On day 5 after incubation, the cells were treated with BrdU (BD Bioscience, San Jose, CA, USA) so that levels of the DNA newly synthesized for the previous 24 hrs in the responder cells were determined by measuring absorbance at 370 nm on a VERSAmax™ microplate reader (Molecular Devices Co., Sunnyvale, CA, USA). The results are shown in FIG. 10. As can be seen in FIG. 10, the proliferation was induced in the PHA-L(H)-stimulated unrelated peripheral blood monocytes (A+H) whereas umbilical cord blood-derived mesenchymal stem cells did not stimulate the responder cells, thus resulting in no induction of cell proliferation (hUCB-MSC+A). Particularly, the umbilical cord blood-derived mesenchymal stem cells were observed to have greater inhibitory effects on the proliferation of the responder cells when they were cultured in the CMH condition than in the typical condition. These data indicate that the umbilical cord blood-derived mesenchymal stem cells cultured in the CMH are less apt to be immunogenic than are those cultured in a typical condition.

When applied to the situation in which the immune response was induced by a reaction between the responder cells (A) and the stimulator cells (B), i.e., (A+B), or by the artificial stimulation of the responder cells (A) with PHA-L, i.e. , (A+H), the umbilical cord blood-derived mesenchymal stem cells cultured in the CMH condition were observed to suppress the proliferation of the responding peripheral blood monocytes more greatly than did those cultured in the typical condition. Similar results were obtained with umbilical cord blood-derived mesenchymal stem cells obtained from different sources although there was a difference to some, but slight degree. These data demonstrate that a CMH culture condition is advantageous over typical conditions in terms of the suppression of immunogenicity.

After the mesenchymal stem cells were reacted in the same manner as described above, PGE₂ (prostaglandin E₂), an immunosuppressant, released therefrom was analyzed using a PGE2 ELISA kit (Cayman, Ann Arbor, MI, USA) according to the protocol of the manufacturer. The cultures from the MLR were used as specimens.

Standards necessary for ELISA assay were prepared to have a maximum density of 1,000 pg/mL, with a minimum density of 7.8 pg/mL serially half-diluted from the maximum. Each of the standards and the culture supernatants of the test group was added in an amount of 50 μί to each well of PGE₂ capture antibody- coated plates. Then, 50 LL of the PGE₂ AchE tracer and 50 of a primary antibody were added to each well, followed by incubation at 4 °C for 18 hrs. The plates were washed five times with a wash buffer, and 200

of Ellman's reagent (included within the kit), was added to each well, followed by the addition of 5 of the tracer per well. The plates were incubated for 60 to 90 min in a dark condition, and absorbance was read at 450 nm.

The results are given in FIG. 11. As can be seen in FIG. 11 , the umbilical cord blood-derived mesenchymal stem cells were observed to release PGE₂ in an approximately 3.7-fold greater amount when cultured in the CMH condition than in the typical condition. Similar results were obtained with different umbilical cord blood-derived mesenchymal stem cells. These data demonstrate that the umbilical cord blood-derived mesenchymal stem cells cultured in the CHM condition were more immunosuppressant than those cultured in a typical condition.

EXAMPLE 8: In Vitro Assay for Ability of Umbilical Cord Blood- Derived Mesenchymal Stem Cells to Release Cytokines According to Culture Condition Effects of culture conditions on the ability of umbilical cord blood-derived mesenchymal stem cells to release cytokines were assayed by measuring Tsp-2 released during the differentiation of the umbilical cord blood-derived mesenchymal stem cells into chondrocytes.

Umbilical cord blood-derived mesenchymal stem cells were cultured in a typical condition (control) and in a CMH condition in the same manner as in Example 3. When reaching 80 to 90% confluency, they were detached by treatment with trypsin. After centrifugation, the cell pellets were washed with high glucose DMEM containing 40 μg/mL L-proline, 0.6

dexamethasone, 50 μg/mL ascorbic acid, and 100

sodium pyruvate, to completely remove FBS from the cells. The umbilical cord blood-derived mesenchymal stem cell pellets obtained again by centrifugation were suspended at a density of 2.0x 10⁵ cells/400 iL, and placed in an aliquot of 400

in 15 mL conical tubes. After centrifugation at 550*g for 5 min, the tubes were so very loosely closed. The tubes were incubated for 24 hrs while being placed upright in a rack. Once a pellet was formed, the supernatant was collected and analyzed for the level of Tsp-2 using a Tsp-2 assay kit (R&D systems, USA).

The results are given in FIG. 12. Tsp-2 is a factor accounting for the titer of umbilical cord blood-derived mesenchymal stem cells for use as a cartilage regenerating agent. Cells that released a higher level of Tsp-2 were evaluated to regenerate cartilage more effectively. As is apparent from the data of FIG. 12, all of four different umbilical cord blood-derived mesenchymal stem cells released higher levels of Tsp-2 in the CMH condition than in the typical condition.

Taken together, the data obtained above indicate that the umbilical cord blood-derived mesenchymal stem cells cultured in the CMH condition have excellent potential of differentiating into chondrocytes and are thus useful as a cartilage regenerating agent.

EXAMPLE 9: Preparation of Aggregates of Umbilical Cord Blood- derived Mesenchymal Stem Cells Cultured in a CMH Condition

Mesenchymal stem cells derived from umbilical cord blood were cultured in a CMH condition, and then allowed to form aggregates by using a hanging drop method to obtain umbilical cord blood-derived mesenchymal stem cell aggregates.

Specifically, human umbilical cord blood-derived mesenchymal stem cells derived from two different sources (MSC #1 and #2) stored in a frozen state were thawed, and cultured at 37 °C in a-MEM medium (Invitrogen, USA) supplemented with 10% FBS under a 5% C0₂ condition in a Forma Series II Water- Jacketed C0₂ incubator (Thermo Scientific Inc.). When the cells were grown to 80 to 90% confluency, they were separated into single cells by treatment with trypsin. The mesenchymal stem cells thus obtained were inoculated in a concentration ranging from 5,000 to 8,000 cells/cm² into a-MEM medium (supplemented with 10% FBS) where 1.8 mM calcium and 1 mM magnesium were further added thereto. The cells were cultured in a 3% (v/v) oxygen (hypoxic) condition, with passages upon 80 to 90% confluency.

Meanwhile, an aggregate formation medium was prepared by adding 20% KnockOut™ SR (Invitrogen), 0.1 nmol/L β-mercaptoethanol, 1 % non-essential amino acid (Invitrogen) and 50 μg/mL gentamycin to DMEM/F-12 medium (Invitrogen).

After 2 passages, the mesenchymal stem cells were separated into single cells by treating with trypsin. The cells were counted, inoculated at l x lO³ cells/20 ΐ, (IK), l lO⁴ cells/20 μΐ, (10K) and 2>< 10⁴ cells/20 μΐ, (20K) into the media, and then the mesenchymal stem cells were placed on a 100 i>(mm) plate with 20 μΐ, drops. Subsequently, PBS was added to the plate in order to prevent the drops from drying, and the plate was turned upside down so that the cells were cultured against gravity to prepare aggregates. The cells were cultured in an incubator at 37 °C for 2 days under atmospheric condition with 5% C0₂.

COMPARATIVE EXAMPLE 1: Preparation of Aggregates of Umbilical Cord Blood-derived Mesenchymal Stem Cells Cultured in a Typical Condition

The procedures of Example 9 were repeated, except that neither calcium nor magnesium were added, and the mesenchymal stem cells were cultured in a 21 % (v/v) oxygen (normoxia) condition instead of a hypoxic condition, to prepare umbilical cord blood-derived mesenchymal stem cell aggregates.

EXPERIMENTAL EXAMPLE 1: Comparison of the Degree of Formation of Umbilical Cord Blood-derived Mesenchymal Stem Cell Aggregates Depending on Culture Conditions

The umbilical cord blood-derived mesenchymal stem cell aggregates prepared in Example 9 and Comparative Example 1 were observed using an ECLIPSE TE2000-U microscope (Nikon Co., Kanagawa, Japan).

Microscopic photographs (40x original magnification) depicting the mesenchymal stem cell aggregates. As can be seen from FIG. 13, the aggregates cultured under the typical condition tend to clump and becomes irregular in shape as they get bigger. In contrast, the aggregates cultured under the CMH condition could maintain spherical shapes well and form a dense and rigid structure as compared to the aggregates cultured under the typical condition. The same result was obtained when aggregates prepared from umbilical cord blood-derived mesenchymal stem cells derived from two different sources were compared. Therefore, it can be concluded that it is preferable to culture the cells in a CMH condition to prepare aggregates.

EXPERIMENTAL EXAMPLE 2: Comparison of Immunogenicity of Umbilical Cord Blood-derived Mesenchymal Stem Cell Aggregates Depending on Culture Conditions

In order to compare immunological properties of umbilical cord blood- derived mesenchymal stem cell aggregates depending on culture conditions, the following experiment was conducted.

For negative control, umbilical cord blood-derived mesenchymal stem cells cultured (2* 10⁴ cells) in the presence of 10 g/mL mitomycin C (Sigma-Aldrich, St Louis, MO, USA) in a typical condition and in a CMH condition, and umbilical cord blood-derived mesenchymal stem cell aggregates (IK, 10K and 20K) prepared Example 9 and Comparative Example 1 were prepared. In case of the aggregates prepared in Example 9 and Comparative Example 1, the concentrations of the cells equally adjusted by adding 20, 2 and 1 aggregates per well for IK, 10K and 20K samples, respectively. Meanwhile, l x lO⁵ cells of responder cells (peripheral blood mononuclear cells (expressed as "A"); ALLCELLS, Emeryville, CA) were treated with 10 μg/mL PHA-L (leucoagglutinin) (expressed as "H"; Roche Diagnostics GmbH, Mannheim, Germany) to prepare a positive control. A test group was prepared by treating the negative control with 1 * 10⁵ cells of peripheral blood mononuclear cells (A).

The negative control, positive control and test group were incubated for 5 days in 96-well plates, and the proliferation and colony formation of the responder cells were observed under a microscope. The cells were treated with BrdU (BD Bioscience, San Jose, CA, USA) so that levels of the newly synthesized DNA of the responder cells for 24 hrs were determined by measuring absorbance at 370 nm on a VERSAmax™ microplate reader (Molecular Devices Co., Sunnyvale, CA, USA).

The results are shown in FIG. 14. As can be seen from FIG. 14, the proliferation was induced in the PHA-L-stimulated peripheral blood mononuclear cells (A+H), whereas umbilical cord blood-derived mesenchymal stem cells and aggregates thereof did not stimulate the responder cells, thus resulting in no induction of cell proliferation. The aggregates (10K and 20K) were observed to have less immunogenicity as compared to mesenchymal stem cells, and particularly, the aggregates which were cultured in the CMH condition showed more reduced immunogenicity than the aggregates which were cultured in the typical condition. Also, it was found that there exists an inverse relationship between the immunogenicity among the aggregates and the size of the aggregates. These data demonstrate that the mesenchymal stem cell aggregates show lower immunogenicity than the mesenchymal stem cells, and the aggregates cultured in the CHM condition show more reduced immunogenicity than those cultured in the typical condition, and therefore, the aggregates cultured in the CMH condition is advantageous as a cell therapeutic agent.

EXPERIMENTAL EXAMPLE 3: Comparison of Immunosuppresion of Umbilical Cord Blood-derived Mesenchymal Stem Cell Aggregates Depending on Culture Conditions

In order to compare immunosuppresion of umbilical cord blood-derived mesenchymal stem cell aggregates depending on culture conditions, the following experiment was conducted.

For negative control, umbilical cord blood-derived mesenchymal stem cells cultured (2x l 0⁴ cells) in the presence of 10 μg/mL mitomycin C (Sigma-Aldrich, St Louis, MO, USA) in a typical condition and in a CMH condition, and umbilical cord blood-derived mesenchymal stem cell aggregates (IK, 10K and 20K) prepared in Example 9 and Comparative Example 1 , l x l O⁵ cells of responder cells (A), and l x lO⁵ cells of stimulating cells (unrelated peripheral blood mononuclear cells (expressed as "B"); ALLCELLS, Emeryville, CA) were prepared. In case of the aggregates prepared in Example 9 and Comparative Example 1 , the concentrations of the cells were made equal by adding 20, 2 and 1 aggregates per well for I , 10K and 20K samples, respectively. Meanwhile, a mixture of l x lO⁵ cells of responder cells (A) and l x lO⁵ cells of stimulating cells (B), and responder cells (A) were treated with PHA-L (H) prepared as a positive control. A test group was prepared by treating the negative control with l x lO⁵ cells of responder cells (A) and l x lO⁵ cells of stimulating cells (B).

The results are shown in FIG. 15. As shown in FIG. 15, when the umbilical cord blood-derived mesenchymal stem cells and aggregates cultured in the typical condition were added to the well where the immune response was induced by a reaction between the responder cells (A) and the stimulating cells (B), it is observed that the proliferation of the responder cells was not significantly inhibited. In contrast, when the umbilical cord blood-derived mesenchymal stem cells and aggregates cultured in the CMH condition were added, the proliferation of the responder cells was suppressed by 50% or more as compared to those cultured in the typical condition. These data demonstrate that a CMH culture condition is advantageous over typical conditions in terms of the immunosuppression.

EXPERIMENTAL EXAMPLE 4: Comparison of the Cytokine Release Ability of Umbilical Cord Blood-derived Mesenchymal Stem Cell Aggregates Depending on Culture Conditions

In order to investigate the therapeutic effectiveness in cartilage regeneration and treatment of pulmonary diseases, umbilical cord blood-derived mesenchymal stem cell aggregates cultured in a typical and in a CHM condition were assayed by measuring Tsp-2 and VEGF, which are known as cytokines to be associated with cartilage regeneration therapy and the treatment of pulmonary diseases, respectively.

Specifically, umbilical cord blood-derived mesenchymal stem cells cultured on a 60 plate coated with a cell adhesion material, e.g., plasma or corona, in the presence of 10 μg/mL mitomycin C (Sigma- Aldrich, St Louis, MO, USA) in a typical condition, the umbilical cord blood-derived mesenchymal stem cell aggregates (IK, 1 OK and 20K) prepared in Example 9 and Comparative Example 1 were inoculated in a concentration of 3>< 10⁵ cells/well into a serum- free MEM-a medium and then cultured for two days. In case of the aggregates prepared in Example 9 and Comparative Example 1, the concentrations of the cells were made equal by adding 300, 30 and 15 aggregates per well for IK, 10K and 20K samples, respectively. After centrifugation, the supernatant was collected and analyzed for the level of each cytokine using Tsp-2 and VEGF assay kits (R&D systems, USA). The results are shown in FIG. 16. As can be seen from FIG. 16, umbilical cord blood-derived mesenchymal stem cell aggregates cultured in the CMH condition released higher levels of cytokines as compared to those cultured in the typical condition. Also, the difference between these two conditions markedly became larger as the size of the aggregates increased.

Taken together, the data obtained above indicate that the umbilical cord blood-derived mesenchymal stem cell aggregates cultured in a CMH condition is advantageous over those cultured in a typical condition in terms of their effectiveness in cartilage regeneration and treatment of pulmonary diseases.

Claims

What is claimed is:

1. A method for preparing mesenchymal stem cell aggregates, which comprises the steps of:

(1) culturing mesenchymal stem cells in a medium containing calcium in a concentration of from 2.1 to 3.8 mM and magnesium in a concentration of from 1.0 to 3.0 mM under a hypoxic condition with a 2 to 5% of oxygen concentration; and

(2) forming aggregates by culturing the mesenchymal stem cells thus obtained in a medium containing serum replacement instead of fetal bovine serum.

2. The method of claim 1, wherein the mesenchymal stem cells are derived from umbilical cord blood, bone marrow, lipid, muscle, skin, amniotic fluid, umbilical cord, or teeth.

3. The method of claim 1, wherein the medium of Step (1) is selected from the group consisting of a Dulbecco's modified eagle medium (DMEM), a minimal essential medium (MEM), an a-MEM, a McCoys 5A medium, an eagle's basal medium, a CMRL (Connaught Medical Research Laboratory) medium, a Glasgow MEM, a Ham's F-12 medium, an IMDM (Iscove's modified Dulbecco's medium), a Leibovitz's L-15 medium, an RPMI (Roswell Park Memorial Institute) 1640 medium, a medium 199, and a Hank's medium 199.

4. The method of claim 3, wherein the medium of Step (1) comprises 5 to 30% of fetal bovine serum.

5. The method of claim 3, wherein the medium of Step (1) does not comprise fetal bovine serum, but a serum replacement.

6. The method of claim 1, wherein the medium of Step (1) is an a-MEM supplemented with 5 to 30% of fetal bovine serum (FBS), where 0.3 to 2.0 mM of calcium and 0.2 to 2.2 mM of magnesium are added.

7. The method of claim 1, wherein the medium of Step (2) is selected from the group consisting of a Dulbecco's modified eagle medium (DMEM), a minimal essential medium (MEM), an a-MEM, McCoys 5A medium, an eagle's basal medium, a CMRL (Connaught Medical Research Laboratory) medium, a Glasgow MEM, a Ham's F-12 medium, an IMDM (Iscove's modified Dulbecco's medium), a Leibovitz's L-15 medium, an RPMI (Roswell Park Memorial Institute) 1640 medium, a medium 199, and a Hank's medium 199.

8. The method of claim 1, wherein the culture of the mesenchymal stem cells in Step (2) is carried out in a hanging drop placed against gravity.

9. The method of claim 1 , wherein the culture of the mesenchymal stem cells in Step (2) is carried out by suspension culture employing a low-attachment culture dish.

10. The method of claim 1, wherein the culture of mesenchymal stem cells in Step (2) is carried out by employing a three-dimensional bioreactor.

11. Mesenchymal stem cell aggregates prepared by the method of claim 1.

12. A cell therapeutic agent comprising the mesenchymal stem cell aggregates of claim 11.

13. The cell therapeutic agent of .claim 12, wherein the cell therapeutic agent finds applications in regeneration or protection of adipocytes, osteocytes, chondrocytes, myocytes, neurocytes, cardiomyocytes, hepatocytes, islet beta cells, vascular cells, or pneumocytes.

14. The cell therapeutic agent of claim 12, wherein the cell therapeutic agent is useful for one selected from the group consisting of treatment of pulmonary diseases; suppression or treatment of lung disease-induced inflammation; regeneration of pulmonary tissues; and suppression of pulmonary fibrosis.

15. The cell therapeutic agent of claim 12, wherein the cell therapeutic agent is applied to cartilage regeneration.

16. The cell therapeutic agent of claim 12, wherein the cell therapeutic agent is applied to therapy of autoimmune diseases, or graft-vs-host diseases.

17. A use of the mesenchymal stem cell aggregates prepared by the method of claim 1 in the manufacture of a medicament for: (1) regeneration or protection of adipocytes, osteocytes, chondrocytes, myocytes, neurocytes, cardiomyocytes, hepatocytes, islet beta cells, vascular cells, or pneumocytes; (2) one selected from the group consisting of treatment of pulmonary diseases, suppression or treatment of lung disease-induced inflammation, regeneration of pulmonary tissues, and suppression of pulmonary fibrosis; (3) cartilage regeneration; or (4) therapy of autoimmune diseases, or graft-vs-host diseases.