KR101459814B1 - Undifferentiated mesenchymal stem cells with higher self-renewal and osteogenic potential and method for preparing the same - Google Patents

Abstract

The present invention relates to an undifferentiated mesenchymal stem cell having an accelerated cell proliferative potency, autotophoresis ability and increased osteocyte differentiation ability, and a method for producing the same. More specifically, the present invention relates to a mesenchymal stem cell cultured in a culture medium containing human umbilical cord blood serum To mesenchymal stem cells and a method for producing the same. The present invention also relates to a composition for inducing bone differentiation for differentiating mesenchymal stem cells containing human umbilical cord blood serum and bone formation inducer into an osteocyte and a method for inducing bone differentiation using the same. The present invention also relates to a composition for the treatment of bone metabolic diseases including undifferentiated mesenchymal stem cells having accelerated cell proliferation and self-renewal ability and increased osteoclast differentiation ability.

Description

[0001] The present invention relates to an undifferentiated mesenchymal stem cell having increased self-renewal ability and osteoclast differentiation ability, and a method for preparing the same,

The present invention relates to an undifferentiated mesenchymal stem cell having increased self-renewal ability and increased osteocyte differentiation ability, and more particularly, to a mesenchymal stem cell obtained by culturing in a medium containing human umbilical cord blood serum, Cells and a method for producing the same. The present invention also relates to a composition for inducing bone differentiation for differentiating mesenchymal stem cells containing human umbilical cord blood serum and bone formation inducer into an osteocyte and a method for inducing bone differentiation using the same. The present invention also relates to a composition for the treatment of bone metabolic diseases including undifferentiated mesenchymal stem cells having accelerated cell proliferation and self-renewal ability and increased osteoclast differentiation ability.

Human mesenchymal stem cells (MSCs) are non-invasive, sticky adult stem cells that are free from cancer and ethical problems that are generally a problem in embryonic stem cells, It is possible to differentiate into various cells such as cells, cartilage cells, cardiac cells, muscle cells and neurons.

The mesenchymal stem cells have a common cell surface phenotype and are positive for CD90 (Thy-1), CD166 (SB10 / ALCAM), CD73 (SH3), CD105 (SH2, endoglin) CD45, HLA-DR or B7. While these commonalities are common, mesenchymal stem cells have observed a considerable degree of heterogeneity in morphology, growth, and differentiation potential. On the other hand, in vitro proliferated mesenchymal stem cells have been used for various cellular therapy for the regeneration of damaged tissues in cardiovascular, nervous, and musculoskeletal systems, while the research on signals regulating the proliferation and differentiation of mesenchymal stem cells is still ongoing. Has been used in the attempt of. In addition, it has been used for the purpose of reducing graft-versus-host disease or inhibiting graft-versus-host disease in facilitating hematopoietic cell transplantation. As clinical trials have increased, there has been a growing interest in optimizing the stability and functionality of in vitro cultured mesenchymal stem cells for clinical applications.

Traditionally, the classification and culture of mesenchymal stem cells has been using fetal bovine serum (FBS). However, safety issues have been raised since FBS can cause immune reactions such as Arthus' reaction, inappropriate reactions such as reactions to heterologous antigens occurring in patients, and the like. And FBS or animal serum have a relatively high risk of prion diseases that cause microbial infections, viral or bacterial infections, or mad cow disease. Therefore, an alternative culture medium capable of supporting the mesenchymal stem cells in an appropriate and stable manner while being able to adequately and stably replace the FBS is being developed, and it has been proposed that cultured medium containing many of the already identified serum or human or allogeneic human- Studies on mesenchymal stem cells have been conducted.

Therefore, the present inventors have attempted to develop an alternative culture medium capable of appropriately and stably replacing FBS and equally supporting mesenchymal stem cells. As a result, it has been found that the umbilical cord blood serum (CBS) The present inventors have completed the present invention by confirming that the mesenchymal stem cells grown are unique characteristics such as high self-renewal ability and increased bone differentiation ability.

One object of the present invention is to provide a compound having the ability to express Oct4; Has enhanced cell proliferation and self-renewal ability; Have the ability to express osteopontin, osteocalcin and ALP (alkaline phosphatase); And nestin-expressing ability of the undifferentiated mesenchymal stem cells.

Another object of the present invention is to provide a method for producing the mesenchymal stem cells, which comprises culturing the mesenchymal stem cells in a culture medium containing human umbilical cord blood serum.

Another object of the present invention is to provide a method for inducing differentiation of mesenchymal stem cells into osteocytes, which comprises culturing mesenchymal stem cells in a culture medium containing human umbilical blood serum and bone formation inducing substance.

Another object of the present invention is to provide a composition for inducing bone differentiation for differentiating mesenchymal stem cells into osteocytes, which comprises human umbilical cord blood serum and an osteogenesis inducer as an effective ingredient.

Another object of the present invention is to provide a composition for treating bone metabolic diseases comprising the mesenchymal stem cells.

In one embodiment, the present invention relates to undifferentiated mesenchymal stem cells that exhibit the following characteristics:

(a) has the ability to express Oct4;

(b) has enhanced cellular proliferative and self-renewing ability;

(c) has the ability to express osteopontin, osteocalcin and ALP (alkaline phosphatase); And

(d) nestin-expressing ability.

Oct4, which has been shown to express undifferentiated mesenchymal stem cells of the present invention, is a transcription factor originally expressed in embryonic stem cells, and plays a role in preventing cell differentiation, It is known as a marker specific for embryonic stem cells that are capable of differentiating into differentiation at the onset of natural differentiation (Donovan, PJ, Nature Genet., 29: 246, 2001; Pesce, M. and Scholer, HR, Stem Cells, 19 : 271, 2001). Therefore, the present inventive mesenchymal stem cells express Oct4 which is a marker of embryonic stem cell that is capable of differentiating. The result shows that the undifferentiated mesenchymal stem cells according to the present invention are more likely to develop embryonic stem cells Of the genetic status.

In addition, the undifferentiated mesenchymal stem cells of the present invention have enhanced cell proliferation and self-renewal ability. The undifferentiated mesenchymal stem cell of the present invention proliferates with a shorter divalent time (Figs. 1A and 1B) than that of mesenchymal stem cells proliferated in CBP (cord blood plasma) or FBS (Fig. 1A and 1B) Proliferation was maintained during 10 passages (Figure 1C). This accelerated mesenchymal stem cell proliferation is associated with increased cell cycle activity, as demonstrated by high BrdU entry, and is associated with a high expression of cyclin D2, the cell cycle regulator (Fig. 2A And FIG. 2B).

The osteopontin, osteocalcin and ALP (alkaline phosphatase) genes, which are found to express the undifferentiated mesenchymal stem cells of the present invention, are known to be originally differentiation markers specific to bone cells. Therefore, the result that the mesenchymal stem cells of the present invention already express osteopontin, osteocalcin and ALP (alkaline phosphatase) before differentiation suggests that the undifferentiated mesenchymal stem cells according to the present invention exhibit an intermediate Unlike lobular stem cells, which actively accepts gene-specific gene expression and is particularly sensitized by bone differentiation. In this regard, the undifferentiated mesenchymal stem cells of the present invention exhibited high osteocalcin promoter activity at the growing stage because the mesenchymal stem cells of the present invention were already sensitized to bone differentiation and thus the basal state and transcription promoted osteocalcin it can be seen that the osteocalcin promoter activity is increased (Fig. 4).

In addition, the nestin gene, which has been found to express the undifferentiated mesenchymal stem cells of the present invention, is originally known as a neural cell-specific differentiation marker. Therefore, the result that the mesenchymal stem cell of the present invention expresses imestin (nestin) before differentiation indicates that the undifferentiated mesenchymal stem cells according to the present invention are different from the mesenchymal stem cells (Fig. 5A and 5B). ≪ / RTI >

In addition, the undifferentiated mesenchymal stem cells of the present invention are characterized by having a colony forming unit-fibroblast (CFU-F) colony forming ability of 20% or more (FIG. 2C). Since mesenchymal stem cells are similar to fibroblasts while forming colonies, they are called CFU-F (colony forming unit-fibroblastic) and CFU-F is a colony formed from a single mesenchymal stem cell Proved.

In addition, the undifferentiated mesenchymal stem cells of the present invention are characterized in that β-catenin or a yes-associated protein (YAP) expression level is enhanced (FIG. 7B). It is known that β-catenin expression inhibits differentiation into adipocytes and enhances osteoclast differentiation, and YAP, a yeast-related protein that binds to TAZ-related transcription factor and also binds to 14-3-3, . Therefore, the above-mentioned result that the mesenchymal stem cells of the present invention highly express beta -catenin or YAP, indicates that the undifferentiated mesenchymal stem cells according to the present invention exhibit a gene-specific gene expression unlike ordinary mesenchymal stem cells And that it is sensitized by osteoclast differentiation.

In addition, the undifferentiated mesenchymal stem cells of the present invention are characterized in that the receptors of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) exhibit high phosphorylation (FIG. 7A ). Platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors are expressed in mesenchymal stem cells, and these two growth factors are known to affect the growth and differentiation of mesenchymal stem cells. The undifferentiated mesenchymal stem cells according to the present invention are characterized by high phosphorylation of PDGF and EGF receptor, unlike general mesenchymal stem cells.

In another aspect, the present invention provides a method for producing mesenchymal stem cells, comprising culturing mesenchymal stem cells in a culture medium containing human umbilical cord blood serum.

In the present invention, the medium containing the human umbilical cord blood serum may further include any one of the following features:

(a) a medium containing human umbilical cord blood serum heat-treated at 40 占 폚 to 60 占 폚;

(b) a medium containing carbon-adsorbed human umbilical cord blood serum; And

(c) A medium containing a human umbilical cord blood serum fraction having a molecular weight of 10 KD or more.

The human umbilical cord blood serum used in the present invention includes a case where the serum of each individual or multiple donors is pooled. Such human umbilical cord blood serum is preferably contained in an amount of 5 to 15% of the total culture medium composition, but is not limited thereto and may be appropriately modified by those skilled in the art.

The present inventors have confirmed that similar effects are obtained when pooling the umbilical cord blood of an individual and at the same time the effect of heat inactivation of these pooled umbilical cord blood cells or of charcoal adsorption and molecular weight fractionation As a result of the analysis, it was confirmed that the pooling effect was the same as that of each donor, and it was confirmed that the effect was further increased by heat treatment. In addition, when the fractions were fractionated at a molecular weight of 10 KD or less, their activities were lost, whereas when fractions of 10 KD or more were retained, the original activity was maintained. Thus, the active component in the umbilical cord blood was in a fraction of a macromolecular weight of 10 KD or more (Figs. 8C to 8E).

In another aspect, the present invention provides a composition for inducing bone differentiation for differentiating mesenchymal stem cells containing human umbilical cord blood serum and bone formation inducer into an osteocyte, and a composition for inducing differentiation into bone cells using the composition ≪ / RTI >

The human umbilical cord blood serum used in the present invention includes a case where the serum of each individual or multiple donors is pooled. Such human umbilical cord blood serum is preferably contained in an amount of 5 to 15% of the total culture medium composition, but is not limited thereto and may be appropriately modified by those skilled in the art.

In the present invention, the bone formation inducing substance may preferably be? -Glycerophosphate, dexamethasone or ascorbic acid, but is not limited thereto.

The present inventors induced human bone marrow hematopoietic serum by adding a serum and a human umbilical cord blood serum to DMEM containing 10 nM dexamethasone, 0.2 mM ascorbic acid and 10 mM? -Glycerophosphate, (Fig. 4B). This suggests that ALP, which is a bone cell differentiation marker, is induced at a remarkably high efficiency from the initial time point, which promotes bone morphogenesis of MSC in CBS medium composition (Fig. 4B).

In another aspect, the present invention provides a composition for treating bone metabolic diseases comprising the mesenchymal stem cells. Since the mesenchymal stem cells of the present invention have an increased bone differentiation ability, they can be used as a composition for bone tissue formation by administration to an individual having a bone metabolic disease.

In the present invention, "bone metabolic disease" refers to a disease that results in a physiological pathological condition due to excessive bone resorption and absorption. Examples of bone metabolic diseases include, but are not limited to, bone metastatic lesions in which tumors such as osteoporosis, breast cancer or prostate cancer have metastasized to the bone, primary bone tumors, rheumatic or degenerative arthritis, periodontal disease, Alveolar bone resorption disease, inflammatory bone resorption disease and Paget's disease, and preferably refers to osteoporosis, inflammatory alveolar bone disorder, rheumatoid arthritis, but is not limited thereto.

Since the undifferentiated mesenchymal stem cells of the present invention cultured in vitro from human umbilical cord blood serum have high self-renewal ability and increased bone differentiation ability, they can be useful for the treatment of bone metabolic diseases and the like.

FIG. 1 shows the extracellular growth rate of MSC according to different culture conditions. (A) was adhered to a medium containing 5, 10, 15% FBS, CBS or CBP at a density of 5,000 cells / cm ² and cultured for 3 days (B) shows the result of measuring the doubling time by collecting the MSC cultured at a density of 2,500 cells / cm ² after the lapse of a predetermined time, and FIG. (C) shows the proliferation proportional value obtained from three independent serogroups in relation to the CBS effect according to the MSC passage culture in terms of the mean ± standard error, and (D) shows the cultivation in two kinds of homologous human adult sera (HS1, HS2) The results of the WST-1 analysis of the growth of MSCs are shown as the mean ± standard error from the results of three experiments.
Figure 2 shows that MSCs cultured in CBS exhibit high colony forming ability and cell cycle activity. Specifically, (A) shows the results of the cell cycle activity of the MSC cultured in FBS, CBS, and CBP as an amount of BrdU after 24 hours of treatment with BrdU, Shows the results of a reverse transcription-polymerase chain reaction comparing the amount of the gene of the cell cycle regulator expressed in MSC cultured in CBS or FBS, and (C) shows the result of CFU- F frequency rate, the mean ± standard error obtained from three experimental results.
FIG. 3 shows the cell phenotype of MSCs cultured in CBS or FBS. FIG. 3 (A) shows the result of analysis of cell surface markers by flow cytometry according to each medium composition (black line indicates the control group (Bars, 2 mm), (C) represents the forward scattering of the flow cytometry (FSCs), (B) ), Indicating that the CBS group has a smaller cell size.
Fig. 4 shows the increased bone-differentiation capacity of MSC cultured in CBS medium, (A) quantifying the amount of mineralized nodules of MSCs grown in FBS and CBS, (B) quantifying alkaline phosphatase (D0) and the amount of osteoclone, osteopontin, osteocalcin, and ALP were measured by reverse transcription-polymerase chain reaction (RT-PCR) using the p-nitrophenol concentration 7 days after induction (D7). (D) shows that the basal state osteocalcin promoter activity is increased in the CBS group. After the reporter plasmid (6OSE2-luc) for the osteocalcin promoter was introduced into C2C12 cells, the effect of CBS on the promoter activity was examined. After normalization with β-gal activity, the degree of relative transcriptional stimulation of CBS relative to FBS was expressed as mean ± standard error (3 exp, n = 6). (E) shows the effect of CBS on promoting transcription of the osteocalcin promoter mediated by runx2. C2C12 cells were transfected with each reporter (6OSE2-luc or -147OC-luc) and pEF-Runx2 and then subjected to relative luciferase activity And the increase rate of CBS-MSC for the FBS-MSC group.
FIG. 5 shows the effect of CBS on neuronal and adipocyte differentiation of MSCs. (A) and (B) show that cells positive to β-tubulin (red) were identified by immunocytochemistry and quantified (Nuclei are stained with Hoechst (blue) and examined). (D) and (E) were cultured in differentiation induction medium for 7 days, stained with Oil-Red O, and cultured for 3 days. After elution, the amount of fat deposited was quantified by measuring the absorbance at 502 nm. (F) shows the results obtained by reverse transcription-polymerase chain reaction (PCR) for 7 days (D7) after induction of the amounts of ppar-γ and aP2 before differentiation induction (D0).
FIG. 6 shows the effect of the mutual conversion between FBS and CBS in the culture medium on the characteristics of MSC. The MSC cultured in FBS and CBS was changed to a culture medium containing CBS (FC) or FBS (CF) (B) and adipocyte differentiation (C) by comparing cell proliferation (A) and FSC by flow cytometry using WST-1 assay.
Figure 7 shows the differentiated signals cultured in FBS and CBS. (A) shows immunoblotting results showing that MSCs cultured in CBS show higher PDGF and EGF signals, (B) shows CBS or FBS , Western blotting of MSCs cultured on YAP, TAZ, total β-caten, and activated β-catenin (* β-cat).
FIG. 8 shows the effect of homologous umbilical cord blood on the individual differences and the physicochemical characteristics of the active ingredients, and FIG. 8 (A) is a graph showing the effect of the homogenous umbilical cord blood on the mesenchymal stem cell proliferation (Oct-4 and Rex-1) of the mesenchymal stem cells cultured in the serum and umbilical cord blood were analyzed by RT-PCR. (C) After multi-donor pooling of the fractions of umbilical cord blood obtained from the donor, the fraction (MW 10 KD) by heat treatment (55 ° C for 30 min) (HI), carbon adsorption (CS) (D) is a promoting effect of bone tissue differentiation, and (E) is a result of inhibiting adipogenic differentiation of blood (E).

Hereinafter, the present invention will be described more specifically with reference to the following examples. However, the following examples are only illustrative of the present invention and the present invention is not limited thereto.

Example  One. MSC Cultivation of

The process of isolating MSCs from bone marrow obtained with the consent of healthy donors was performed as previously described. Bone marrow mononuclear cells were obtained from the layers separated by Ficoll-Paque ^™ PLUS (GE Healthcare, Uppsala, Sweden) and cultured in DMEM (Dulbecco's modified Eagle's medium) containing 10% FBS. After one week, the unattached cells were discarded and adherent cells were subcultured. To obtain CBS, umbilical cord blood from healthy donors was collected under anticoagulant conditions and allowed to clot for 6-12 hours at room temperature. The supernatant obtained by centrifugation at 2,500 rpm for 30 minutes was sterilized with 0.2 mm pores and stored at -80 ° C until use. The umbilical cord blood umbilical vein (CBP) was obtained by centrifugation at 2,500 rpm for 20 minutes according to the ficoll density separation method. This study was conducted under the approval of Catholic University Ethics Committee.

Example  2. From CBS  Cultured MSC of In vitro growth ability  Confirm

2-1. In subculture  Confirm the effect

MSCs were cultured at a density of 5,000 cells / cm ² in medium containing 5-15% FBS, CBS or CBP to compare the rate of in vitro expansion and proliferation of MSCs grown in medium supplemented with FBS, CBS and CBP . After 3 days, the cells were treated with 0.05% Trypsin / EDTA. Doubling times during subculture were calculated by the following formula. n = log (NH-NI) / log2 (n, number of cells doubled, NI, number of cells at the beginning of culture; NH, number of cells collected at the time of measurement). The doubling time was calculated as t / n (t is the incubation time). Each experiment was measured 3 times and the mean value was obtained and it was repeated 3 times.

MSCs from bone marrow were found to proliferate with shorter times in CBS - containing media compared to CBP or FBS. (35.4 hours in CBS, 83.5 in CBP, 45.2 hours in FBS, p < 0.05) (FIGS. 1A, 1B). Increased cell proliferation in these CBSs was maintained during 10 passages (Figure 1C). This suggests that CBS is effective for cell proliferation at the late stage of MSC.

2-2. WST -1 Confirmation through experiment

For the WST-1 experiment, 10 ml of WST-1 reagent (Roche Diagnostics, Mannheim, Germany) was placed in a 96-well microplate adhered with 1,000 MSCs per well and incubated for 4 hours. The formazan dye isolated from the tetrazolium salt was quantitated with an ELISA reader at a wavelength of 450 nm for 600 nm.

As a result, MSCs were proliferated in the medium containing CBS compared with FBS, but not in the medium containing human serum (HS) (Fig. 1D).

2-3. From CBS  Cultured MSC of Increased  Identification of cell cycle activity

MSCs were treated with 10 mM bromodeoxyuridine (BrdU) for 24 h and analyzed by flow cytometry using BrdU flow kits (BD Pharmingen, San Diego, CA) The amount of BrdU was examined for cell cycle activity.

As a result, accelerated MSC proliferation in CBS medium was associated with increased cell cycle activity, as seen in high BrdU influx (Figure 2A).

Next, the gene amount of the cell cycle regulator expressed in MSCs cultured in CBS or FBS was compared. RNA was separated by a spectrophotometer (Electrofor, Italy) and cDNA was synthesized according to the manufacturer's instructions (SuperScript II, Invitrogen, Carlsbad, Calif.). At this time, GAPDH was standardized. The primers used were prepared as shown in the following table. The PCR was performed at 95 ° C for 3 minutes, followed by 30 cycles of 95 ° C for 30 seconds, 52-55 ° C for 30 seconds, and 72 ° C for 30 seconds.

forward reverse Cyclin D1 5'-ctt cct ctc caa aat gcc ag-3 ' 5'-aga gat gga agg ggg aaa ga-3 ' Cyclin D2 5'-ttc att gca gac acc acc at-3 ' 5'-gcc aga tac cag aag cga ag-3 ' Cyclin D3 5'-tga ttt cct ggc ctt cat tc-3 ' 5'-agc ttg act agc cac cga aa-3 ' P16 5'-cgg aag gtc cct cag aca tc-3 ' 5'-tca tga agt cga cag ctt ccg-3 ' P21 5'-ctc aga gga ggc gcc atg-3 ' 5'-ggg cgg att agg gct tcc-3 ' Cyclin E 5'-agt tct cgg ctc gct cca gga aga-3 ' 5'-tct tgt gtc gcc ata tac cgg tca-3 ' Cyclin 5'-att agt tta cct gga ccc ag-3 ' 5'-CaC aaa ctc tgc tac ttc tg-3 '

As a result of examining the cell cycle control substances, the expression level of cyclin D2 was increased in the CBS group, whereas the cell cycle inhibitors such as cyclin D1, D3, cyclin A, p16 and p21 did not show a significant difference (Fig. 2B ).

2-4. From CBS  Cultured MSC of Increased Colony Formability  ( CFU -F) Confirmation

In order to clarify the characteristics of MSC showing high growth rate, MSC of single cell clone was cultured in each medium and then colony forming ability (CFU-F) was compared. MSC, negative for propidium iodide (PI: 1 μg / ml), was added to a 96-well plate containing 100 μl of the medium using a FACS Vantage SE (Becton Dickinson, Franklin Lakes, NJ) And were confirmed by microscopy. The number of cell clusters consisting of more than 50 cells was cultured for 14 days and counted by staining with crystal violet (Sigma, St. Louis, Mo.).

As a result, the FBS group did not produce any colonies in a 96-well culture dish, which is consistent with previous reports of loss of CFU-F (colony forming unit-fibroblast) following MSC subculture. In contrast, 28% (28 ± 0.4%) of the MSCs grown in CBS formed CFU-F (Figure 2C), which maintains high frequency of CFU-F during MSC proliferation in this condition . Therefore, the MSC of CBS quantitatively accelerates the proliferation and expansion of MSC, and also maintains the colony productivity qualitatively, resulting in high self-productivity and cleavage ability of the propagated MSC.

Taken together, these results indicate that MSC cultured in homologous CBS induces accelerated proliferation of mesenchymal cells with increased self-production.

Example  3. From CBS  Cultured MSC Characterization of

3-1. From CBS  Cultured MSC of Cell marker  Confirm

Flow cytometric analysis of HLA-DR, CD73, CD90, CD34, CD45, CD166, and CD105 was performed to see the differences in the cellular phenotype stages of FBS and CBS cell lines. As monoclonal antibodies against HLA-DR-FITC, CD73-PE, CD90-FITC, CD-34-PE, CD45-PE (BD Pharmingen), and CD166-FITC, CD105-FITC (Serotec, Oxford, UK) After staining, they were analyzed using FACSCalibur (Becton Dickinson) and CellQuest software. For comparison of cell size, PI (-) cells were measured for forward scatter (FSC) and side scatter (SSC), respectively, and were used as an index of cell size and particle size.

As a result, there was no difference in the cell surface markers of MSC (FIG. 3A). However, the CBS group shows lower anterior scattering as shown by flow cell analysis (Fig. 3C).

Next, for ultrastructure analysis, MSC was prefixed with 2% paraformaldehyde / 2.5% glutaraldehyde for 1 hour, and then washed and fixed with 1% osmium tetroxide for 30 minutes. Dehydrated with ethanol / acetone and embedded with Epon 812. Ultrafine slices (60-70 nm) were stained with uranyl acetate and lead citrate for 20 min and 5 min, respectively, and analyzed under JEM-1010 (JEOL, Akishima, Tokyo) electron microscope Respectively.

Electron microscopic analysis showed that the CBS group had more dense nuclear membrane and thin cytoplasm compared to the FBS group (FIG. 3B), which is consistent with the result of FIG. 3C.

3-2. Bone cell Ability to distinguish

The results showed that CBS-containing media had a significant effect on MSC proliferation and self-reproduction. Next, we examined the effect of CBS on multiple differentiation. First, we compared the bone differentiation potential of MSC.

Specifically, bone differentiation was induced by adding 10% FBS or CBS to DMEM containing 10 nM dexamethasone, 0.2 mM ascorbic acid, 10 mM [beta] -glycerophosphate as previously reported. After 2 weeks, the extracellular matrix mineralization was observed by staining with Alizarin red S, followed by the elution of the dye with cetylpyridinium chloride (Sigma) and quantification by measuring the absorbance at 550 nm. Alkaline phosphatase (ALP) activity was obtained by collecting MSCs on the 0, 3, 5, and 7 days after osteogenesis, and then lysing them with manual dissolution buffer (Promega, Madison, WI) -Nitrophenylphosphate (Sigma) as a substrate. ALP activity was converted to the concentration of p-nitrophenol (nanomoles) based on the p-nitrophenol standard solution, where the total protein concentration was normalized.

As a result, 14 days after the differentiation, MSC of CBS was significantly increased in the amount of calcium deposited as compared with FBS, and it was confirmed by alizarin red staining, and the increased amount of mineralized nodules was quantitatively known (FIG. 4A). ALP is induced in the CBS group from the initial time zone as compared with FBS, suggesting that the CBS medium composition promotes osteoblast differentiation (Fig. 4B).

Next, the amount of osteocellular markers runx2, osteopontin, osteocalcin, and ALP was examined by reverse transcription polymerase chain reaction (D7) at 7 days after induction of differentiation induction (D0). RNA was separated by a spectrophotometer (Electrofor, Italy) and cDNA was synthesized according to the manufacturer's instructions (SuperScript II, Invitrogen, Carlsbad, Calif.). At this time, GAPDH was standardized. The primers used were prepared as shown in the following table. The PCR was performed at 95 ° C for 3 minutes, followed by 30 cycles of 95 ° C for 30 seconds, 52-55 ° C for 30 seconds, and 72 ° C for 30 seconds.

forward reverse Osteocalcin 5'-atg aga gcc ctc aca ctc ctc-3 ' 5'-gcc gta gaa gcg ccg ata ggc-3 ' Osteopontin 5'-gtt aaa cag gct gat tct gg-3 ' 5'-ttg acc tca gtc cat aaa cc-3 ' Runx2 5'-ctc act acc ac acct acc tg-3 ' 5'-tca ata tgg tcg cca aac aga ttc-3 ' ALP 5'-tgg agc ttc aga agc tca aca cca-3 ' 5'-atc tcg ttg tct gag tac cag tcc-3 '

As a result, MSCs in the growing stage showed significantly higher expression of osteopontin, osteocalcin and ALP in CBS than FBS, and these genes were expressed in the FBS group after bone formation ( 4C).

Next, in order to investigate the basic expression level of osteocyte genes, the influence of CBS on the osteocalcin promoter after the introduction of the reporter gene into the osteocytic mesenchymal cell, C2C12 cell line, was examined. Specifically, C2C12 cells (ATCC, Manassas, Va.) Were cultured by adding 10% FBS or CBS to DMEM medium containing 100 mg / ml penicillin and streptomycin. To identify the activity of the osteocalcin promoter, p6OSE-2 with 6 Runx2 binding sites and -147 OC-luc containing the endogenous osteocalcin promoter corresponding to 147 bp were used. For osteocalcin, the cells were adhered to a 24-well culture dish 5 x 10 ⁴ per well, and 0.2 μg of reporter plasmid (p6OSE2-luc or -147 OC-luc), 0.05 μg pCMV-β-gal, of pEF-Runx2 was transfected using Lipofectamine Plus reagent (Invitrogen). After 3 hours of DNA addition, the medium was transformed into DMEM containing 10% of each serum. After 24-48 hours, the cells were collected and their activity was measured using the Luciferase Assay System (Promega). The gene transfer efficiency was normalized to? -Galactosidase activity. Bone marrow differentiation was induced by treatment with 300 ng / ml recombinant human BMP-2 (R & D Systems, Minneapolis, Minn.) In the presence of 5% serum. The reporter vectors 6OSE2-luc and 147 OC-luc and pEF-Runx2 plasmids were supplied from Professor Hong Jung-Ho of Korea University.

As a result, the cells cultured in the CBS medium showed high osteocalcin promoter activity (1.8 ± 0.1 folds) at the growing stage, and this phenomenon was also observed in the BMP-2-induced bone differentiation process (FIG. In addition, promoting transcription of the promoter by Runx2 also increased during growth and division (2.0 ± 0.4 folds, FIG. 4E). This suggests that the cells cultured in CBS have already been sensitized to bone differentiation, thereby increasing the basal state and transcription promoted osteocalcin promoter activity.

3-3. Nerve cell Ability to distinguish

To generate nerve cells, a cover glass coated with collagen was adhered to occupy about 60% of the MSC, and the next day, 10 ng / ml bFGF was treated for 24 hours. After washing three times with PBS, the cells were cultured in a serum-free medium containing 25 mM KCl, 2% DMSO, 100 μM BHA, 10 ng / ml bFGF, 2 mM valproic acid and 100 μM phocolin. After 5 hours, the cells were immunostained with? -Tubulin III antibody (Chemicon, Temecula, Calif.).

When MSCs were differentiated into neurons, MSCs with β-tubulin III-positive were observed more frequently in the CBS group (FIGS. 5A and 5B), and in the CBS group before the induction of neural differentiation, higher nestin ) Expression (Fig. 5C).

3-4. Fat cell Ability to distinguish

For the adipocyte differentiation, the cells were cultured so as to occupy a sufficient amount in a culture dish, and then DMEM containing 1 μM dexamethasone, 0.5 mM isobutyl-1-methylxanthine, 100 μM indomethacin and 10 μg / CBS was added and cultured for 1-2 weeks. After staining the cells with 4% formaldehyde and staining with oil red, the fat droplets were observed. After eluting the staining reagent with 4% nonidet 40 / isopropyl alcohol, the adipocytes differentiated with the absorber at the wavelength of 520 nm Were recorded.

In addition, the expression level of PPARγ and aP2, which are adipocyte markers, was examined by reverse transcription-polymerase chain reaction at 7 days (D7) after induction of differentiation induction (D0). RNA was separated by a spectrophotometer (Electrofor, Italy) and cDNA was synthesized according to the manufacturer's instructions (SuperScript II, Invitrogen, Carlsbad, Calif.). At this time, GAPDH was standardized. The primers used were prepared as shown in the following table. The PCR was performed at 95 ° C for 3 minutes, followed by 30 cycles of 95 ° C for 30 seconds, 52-55 ° C for 30 seconds, and 72 ° C for 30 seconds.

forward reverse PPARγ 5'-gct gtg cag gag atc aca ga-3 ' 5'-ggg ctc cat aaa gtc acc aa-3 ' aP2 5'-tca gtg tga atg ggg atg tg-3 ' 5'-gtg gaa gtg acg cct ttc at-3 '

As a result of comparing the differentiation into adipocytes, the number of fat-red adipocytes was significantly decreased in CBS-MSC (Fig. 5D). As a result, the number of fat droplets was reduced by three times (Fig. 5E) and the expression of aP2 gene was very weak (Fig. 5F). Therefore, it can be seen that MSC cultured in CBS is inhibited from differentiation into adipocytes.

In summary, CBS-MSC has a unique multiple differentiation potential, with improved bone differentiation and reduced fat-splitting capacity.

3-5. Of the culture medium FBS Wow CBS Interconversions between MSC Effect on the characteristics of

Previous studies have reported heterogeneity within MSCs and subgroups that differ in their proliferation, size, and differentiation potential. Therefore, we sought to determine whether the differences observed in the different media are due to the selective surviving subgroups in the subculture, or to differences in stimulation in environments with different growth factors. To find the answer to this question, we investigated whether the cell phenotype was restored after the culture conditions of MSC were changed and most of the cells were cultured (ie within 5 days).

Specifically, the MSC cultured in FBS or CBS was changed to a culture medium containing CBS (FC) or FBS (CF), and 5 days later, cell proliferation was assayed by WST-1 assay and FSC was compared by flow cytometry Cell size, and ability to differentiate into adipocytes.

As a result, as shown in FIG. 6A, when the medium was replaced with FBS (CBS) (FC), the proliferation activity of MSC was similar to that of MSC cultured in CBS. On the other hand, substitution of FBS in CBS was similar to the growth of MSCs proliferated in (CF) FBS (Fig. 6A). In addition, the size of the cells in each medium was also reversed when the culture conditions were changed (Fig. 6B). Similarly, the differentiation into adipocytes was reversed when CBS-to-FBS was converted, and when the FBS-to-CBS was changed, the differentiation was inhibited (Fig. 6C). The fact that the cell characteristics were changed in the absence of major cell division suggests that the unique characteristics of FBS and CBS are due to the stimulated effects of specific factors rather than the different MSC groups.

3-6. FBS  Wow CBS Identification of differentiated signals cultured in

Next, we attempted to identify the differentially activated signals in MSCs cultured in CBS and FBS. First, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors are expressed in MSCs. Since these two growth factors are known to affect the growth and differentiation of MSCs, Were compared in each medium condition.

Specifically, a solution containing 0.1% Triton X-100, 1 mM DTT, 15 mM MgCl ₂ , 150 mM NaCl, 20 mM EGTA, 50 mM HEPES (pH 7.5), 25 mM NaF, 1 mM sodium orthovanadate, MSC was treated with lysis buffer containing 50 mM [beta] -glycerophosphate, 1 mM PMSF, postase complex inhibitor (Roche Diagnostics, Mannheim, Germany) and centrifuged at 15,000 rpm for 20 minutes at 4 [deg.] C. 10 μg of PDGFR (Santa Cruz Biotechnology) or EGFR antibody (R & D systems) was reacted at 250 ° C for 2 hours at 4 ° C and reacted with protein A / G agarose (GE Healthcare) for 1 hour. After washing with dissolution buffer, the immunoprecipitated proteins were separated by SDS-PAGE and immunoblotted with phosphotyrosine antibody (p-Tyr, clone 4G10, Upstate).

As a result, MSCs in FBS and CBS similarly expressed PDGF and EGF receptor, but their degree of phosphorylation in the tyrosine was high in the CBS group and hardly observed in the FBS (FIG. 7A). That is, PDGF and EGF receptors showed high phosphorylation in MSCs proliferated in CBS, indicating that PDGF and EGF in CBS give a unique stimulus to MSC.

Next, we investigated the differentiation of the differentiation system from the adipocyte to the osteocyte in the CBS group. Recently, TAZ, a transcriptional activator that has been shown to bind to 14-3-3, has been reported as a mediator that plays a role in converting MSC differentiation from adipocytes into bone cells. Similarly, the expression of [beta] -catenin, which has increased beta -catenin activity or sustained activity by Wnt ligands, inhibits differentiation into adipocytes and enhances osteoclast differentiation. To investigate the similar changes in CBS, we tried to compare the expression levels of these electron regulatory elements in MSCs cultured in CBS and FBS. To investigate the expression levels of TAZ, YAP and β-catenin, 40 ㎍ of lysates were incubated with polyclonal TAZ antibody, total β-catenin antibody (LF-MA0102, LabFrontier, Seoul, Korea) Western blotting was performed using an antibody against catenin (clone 8E7, Upstate, CA, USA). Antibodies against TAZ or YAP were obtained from Dr. Hong Jung-Ho.

As a result, it was found that the total protein amount of β-catenin was increased in the CBS group and the β-catenin in the activated non-phosphorylated form was also increased compared to the MSC cultured in FBS (FIG. 7B). On the other hand, there was no significant difference in the amount of TAZ in the CBS group and in the FBS group, but the expression level of YAP, a yeast-related protein that binds to another TAZ-related transcriptional activator but to 14-3-3, was significantly increased in the CBS group 7B). These results suggest that this is a non-canonical Wnt pathway for accumulation of β-catenin and induction of YAP, which is not accompanied by TAZ changes and explains the transfer of fat-to-bone cell differentiation from MSC cultured in CBS .

Example  4. CBS  For each effect on the origin effect Variation and  Effects on gene expression and physical properties of fractions

4-1. CBS  Variation of the individual effects of each effect

In order to examine whether the hyperplasia effect of allogeneic umbilical cord blood was specific for the individual, cord blood collected from 9 donors were divided into cord blood (CBS) obtained from 8 donors and rats (FBS ) Were compared. The target mesenchymal stem cells were analyzed for cells isolated from two independent donors (MSC # 1, 2). As a result, it was confirmed that a certain effect was observed irrespective of the individual differences (Fig. 8A).

4-2. CBS  Effect of Derived Effect on Gene Expression

The expression of Oct-4 and Rex-1 genes was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in order to confirm whether the culture of mesenchymal stem cells by umbilical cord blood can mimic adult stem cell- -PCR). As a result, it was confirmed that the Oct-4 gene, which is a gene expressed in the embryo stage, was induced to be expressed when cultured in umbilical cord blood (Fig. 8B).

4-3. Cord blood  Physical properties of active ingredients

Next, the results of this study confirm the similar effect when pooling the umbilical cord blood of an individual, and analyze the effects of heat inactivation of these pooled umbilical cord blood, charcoal adsorption and molecular weight fractionation Respectively. Specifically, fractions of umbilical cord blood obtained from 6 donors were pooled and subjected to heat treatment (55 ° C for 30 min) (HI), carbon adsorption (CS) and fraction according to molecular weight (MW 10 KD) were separated by centricon and compared with untreated umbilical cord blood, growth promoting effect, osteogenic differentiation promoting effect, and adipogenic differentiation inhibiting effect were observed.

As a result, it was confirmed that the effect of pooling was the same as that of each donor, and it was confirmed that the effect was further increased by heat treatment. In addition, when the fractions were fractionated at a molecular weight of 10 KD or less, their activities were lost, whereas when fractions of 10 KD or more were retained, the original activity was maintained. Thus, the active component in the umbilical cord blood was in a fraction of a macromolecular weight of 10 KD or more (Fig. 8 CE).

Statistical analysis

All of the above experiments were repeated at least 3 times and the results were expressed as mean ± standard error and analyzed with Student's T-test. P <0.05 was considered statistically significant.

Claims (14)

(I) culturing the mononuclear cell group obtained from bone marrow for 1 week;
(Ii) removing the non-adherent cells after the culture, separating the adherent cells and subculturing the cells; And
(Iii) culturing the adherent cells in a culture medium containing human umbilical cord blood serum.
(a) has the ability to express Oct4;
(b) has a colony forming ability of more than 20% CFU-F (colony forming unit-fibroblast);
(c) has the ability to express osteopontin, osteocalcin and ALP (alkaline phosphatase);
(d) nestin expression ability; And
(e) The ability to differentiate adipocytes is suppressed.
The method of claim 1, wherein the medium containing the human umbilical blood serum is a medium having any one of the following features:
(a) a medium containing human umbilical cord blood serum heat-treated at 40 占 폚 to 60 占 폚;
(b) a medium containing carbon-adsorbed human umbilical cord blood serum; And
(c) A medium containing a human umbilical cord blood serum fraction having a molecular weight of 10 KD or more.
The method of claim 1, wherein said human umbilical blood serum is homogenous or autologous.
The method according to claim 1, wherein the human umbilical cord blood serum is cord blood serum obtained by pooling cord blood serum of a multiple donor.
The method of claim 1, wherein the human umbilical blood serum is contained in an amount of 5 to 15% of the total culture composition.
An undifferentiated mesenchymal stem cell prepared by the method of any one of claims 1 to 5.
[7] The mesenchymal stem cell according to claim 6, wherein the mesenchymal stem cell is an undifferentiated mesenchymal stem cell cultured in a medium containing human umbilical cord blood serum (CBS), wherein the undifferentiated mesenchymal stem cultured in a medium containing fBS (FBS) Wherein the expression level of beta -catenin or yeast-associated protein (YAP) is higher than that of cells.
[7] The mesenchymal stem cell according to claim 6, wherein the mesenchymal stem cell is an undifferentiated mesenchymal stem cell cultured in a medium containing human umbilical cord blood serum (CBS), wherein the undifferentiated mesenchymal stem cultured in a medium containing fBS (FBS) Wherein the stem cell has an osteocalcin promoter activity higher in the growth phase than the cell.
[7] The mesenchymal stem cell according to claim 6, wherein the mesenchymal stem cell is an undifferentiated mesenchymal stem cell cultured in a medium containing human umbilical cord blood serum (CBS), wherein the undifferentiated mesenchymal stem cultured in a medium containing fBS (FBS) Wherein the platelet derived growth factor (PDGF) and epidermal growth factor (EGF) receptors show higher phosphorylation than cells.
[7] The mesenchymal stem cell according to claim 6, wherein the mesenchymal stem cell is an undifferentiated mesenchymal stem cell cultured in a medium containing human umbilical cord blood serum (CBS), wherein the undifferentiated mesenchymal stem cultured in a medium containing fBS (FBS) Wherein the expression level of cyclin D2 (cyclin D2) is higher than that of cells.
A method for inducing differentiation of mesenchymal stem cells into osteocytes, comprising culturing the mesenchymal stem cells of claim 6 in a culture medium containing human umbilical cord blood serum and bone formation inducer.
12. The method according to claim 11, wherein the bone formation inducing substance is selected from the group consisting of? -Glycerophosphate, dexamethasone and ascorbic acid.
A composition for treating bone metabolic diseases comprising the mesenchymal stem cells of claim 6.
A composition for treating neurological diseases comprising the mesenchymal stem cells of claim 6.

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