CN1920010A - Method of separating multipotent adult progenitor cells from umbilical cord blood - Google Patents

Abstract

The invention discloses a method for isolating multipotent adult progenitor cells from umbilical cord blood. Unattached cells were removed, and then replaced with 2-5% fetal bovine serum, human basic fibroblast growth factor (bFGF), human epidermal growth factor (EGF), human platelet-derived growth factor (PDGF-BB) , MCDB-201, ascorbic acid, dexamethasone and ITS additive culture medium to continue adherent culture, subculture, purification and separation. Compared with other methods, the method of the invention is used to separate multipotent adult progenitor cells from umbilical cord blood, and the separation success rate is high, and the obtained cell phenotype, cell cycle and ability to induce differentiation are the same. Therefore, the isolation of pluripotent adult progenitor cells from umbilical cord blood by this method can significantly improve the efficiency.

Description

A method for isolating multipotent adult progenitor cells from umbilical cord blood

technical field

The invention relates to a method for isolating multipotent adult progenitor cells (multipotent adult progenitor cells, MAPC) from umbilical cord blood, in particular to a method for isolating multipotent adult progenitor cells from umbilical cord blood in vitro.

Background technique

As the basic research of tissue engineering, stem cell engineering has an extremely important role and far-reaching impact on future tissue and organ repair and replacement. The key is to obtain stem cells that can differentiate into desired tissues.

Stem cells can be divided into embryonic stem cells and adult stem cells according to the order in which they appear during individual development. Due to the lack of knowledge about the regulatory mechanism of embryonic stem cell differentiation and development, the limited source of embryonic stem cells and the risk of inducing tumors after transplantation, and the research and application of embryonic stem cells involving some ethical issues, embryonic stem cells are still far from clinical application. more distant. With the deepening of stem cell biology research, it has been discovered in recent years that some adult tissues have adult stem cells with multi-lineage differentiation potential. Such cells can not only regenerate specific tissues, but also transform into cells of other tissue systems under certain circumstances. That is, cross-system and even cross-germ layer differentiation and development. The clinical application of adult stem cell transplantation is more realistic because of its rich source (it can be derived from itself) and few ethical issues involved and existing.

At present, most studies on adult stem cells focus on bone marrow-derived mesenchymal stem cells (MSCs), which have been confirmed to differentiate into a variety of human tissue cells, such as osteoblasts, chondrocytes, adipocytes, muscle cells, nerve cells, etc. (Pittenger MF, Mackay AM, Beck SC, et al. Multilineage potential of adult human mesenchymal stem cells. Science, 1999, 284: 143-147.). Its role in hematopoietic regulation, immune regulation, gene therapy and tissue engineering has received more and more attention (Noort WA., Kruisselbrink AB..in't Anker PS. Mesenchymal stem cells promote engraftment of human umbilical cord blood-derived CD34+ cells in NOD/SCID mice. Exp Hematol. 2002; 30: 870-878, Aggarwal S., Pittenger MF. Humanmesenchymal stem cells modulate allogeneic immune cell responses. Blood. 2005; 105: 1815-1822). However, it is difficult to obtain a large number of bone marrow cells for cell transplantation, and the number and differentiation potential of bone marrow MSCs decrease with age (D'Ippolito G, Schiller PC, Ricordi C, et al.Age-related osteogenic potential of mesenchymal stromal stem cells from human vertebral bonemarrow. J Bone Miher Res. 1999; 14: 1115-1122.), so it will be difficult to apply it clinically in the future.

Human umbilical cord blood is rich in hematopoietic stem and progenitor cells. Since the first successful transplantation of umbilical cord blood stem cells in 1988, umbilical cord blood stem cells have been used as an important source of hematopoietic stem cells in clinical practice. Recent studies have shown that in addition to hematopoietic stem cells, there are special non-hematopoietic somatic cell populations similar to bone marrow mesenchymal cells in cord blood (Goodwin HS, Bicknese AR, Chien SN, et al. Multilineage differentiation activity by cellsisolated from umbilical cord blood: expression of bone, fat, and neural markers. Biol Blood Marrow Transplant, 2001, 7(11): 581-8; Lee OK, Kuo TK, Chen WM, et al. Isolation of multipotent mesenchymalstem cells from umbilical cord blood. Blood.2004, 103(5):1669-75; Kgler G, Sensken S, Airey JA et.al.A Human Somatic Stem Cell from Placental Cord Blood with Intrinsic Pluripotent Differentiation Potential.J Exp Med.2004, 200(2) :123-35), its appearance resembles fibroblasts, does not express hematopoietic cell markers (CD34, CD45, CD14, CD2, CD3, CD15, CD16, CD19, CD24, CD33, CD38, glycophorin A), expresses CD13, Adhesion molecules such as CD29 and CD44, human mesenchymal cell markers SH1 and SH2 are strongly positive, HLA-ABC is positive and HLA-DR is negative. But different from bone marrow mesenchymal stem cells, its ratio is low, about 0.05-2.8 per 10 ⁶ cord blood mononuclear cells, lower than bone marrow mesenchymal stem cells (about 2 per 10 ⁶ mononuclear cells -5, Koc, ON, Lazarus HM. Mesenchymal stem cells: heading into the clinic. BoneMarrow Transplant. 2001; 27: 235-239.), but such cells may be more primitive and more proliferative. At the same time, these cells can express a variety of cytokines, such as SCF, LIF, TGF-1b, M-CSF, GM-CSF, etc., and are superior to bone marrow MSCs in supporting the expansion of CD34 ⁺ cells (Kogler G, RadkeTF , Lefort A, et al. Cytokine production and hematopoiesis supporting activity of cord blood-derived unrestricted somatic stem cells. ExpHematol. 2005; 33(5): 573-83). More importantly, under specific induction conditions, these cells show the potential to differentiate into various tissue cells such as bone, cartilage, fat, nerve, liver, and cardiomyocytes. In animal experiments, it can promote the engraftment of human hematopoietic stem cells, repair tissue damage and improve disease symptoms.

With the establishment of cord blood stem cell banks in recent years, cord blood stem cells have the advantages of strong proliferation and self-renewal ability, weak immunogenicity, etc. The development and application of multipotent adult progenitor cells derived from umbilical cord blood will provide new seed cells for tissue engineering, which has far-reaching significance for stem cell tissue engineering and its application.

At present, most of the isolation of multipotent adult progenitor cells from umbilical cord blood still adopts the method of adherent subculture, but some of them are positively or negatively sorted by immunomagnetic beads through specific surface markers such as STRO-1 and CD45. Due to the low content of these cells and the lack of highly specific surface markers, the success rate of isolation is low, such as Bieback K, Kern S, Kluter H, Eichler H. Critical parameters for the isolation of mesenchymal stem cells from umbilicalcord blood.Stem Cells.2004 ;22(4):625-34. Reported that only 19 of 59 umbilical cord blood samples were successfully separated (28%) under the condition of containing 10% fetal bovine serum without adding growth factors. Domestic reports are similar (29.17%, Zhu Meiling, Hu Yanfen, Wen Guanmei, etc., Isolation and Culture of Human Placental Blood-Mesenchymal Stem Cells, Chinese Journal of Pathophysiology 2004, 20(9): 1743-1744), and the analysis using immunomagnetic beads The selection has the problems of complicated steps and high cost. Therefore, it is urgent to find a simple and easy way to improve the separation effect, so as to provide more sufficient theoretical basis and technical methods for the application of umbilical cord blood multipotent adult progenitor cells in clinical treatment.

Contents of the invention

The technical problem to be solved by the present invention is to provide a method for isolating multipotent adult progenitor cells from umbilical cord blood, so as to overcome the above-mentioned defects in the prior art and meet the needs of tissue engineering cell therapy.

The content of multipotent adult progenitor cells in umbilical cord blood is low, and the success rate of isolation in vitro by the commonly used simple serum-containing adherent culture isolation method is low, and the cells are prone to differentiation under the condition of high serum. At the same time, the composition of serum is complex. Although it contains many components that are beneficial to cells, it also contains components that are harmful to cells. The quality differences between different batches may also make it difficult to standardize experiments and production. not conducive to future applications. However, in previous experiments, we found that simply reducing the serum concentration or performing serum-free culture without adding cell growth additives or cytokines is not conducive to isolation culture. Therefore, we consider that while reducing the serum concentration, adjusting the components of the existing culture reagents and adding appropriate recombinant human cytokines and other growth additives may help reduce the adverse effects of xenogeneic serum and improve the separation effect.

A variety of growth factors have been confirmed to have an effect on the growth of bone marrow MSCs. Since the multipotent adult progenitor cells derived from umbilical cord blood have similar cell phenotype and differentiation potential to bone marrow MSCs, we consider selecting appropriate growth factors that are conducive to the growth of bone marrow MSCs. Factors to promote the growth of multipotent adult progenitor cells derived from umbilical cord blood.

Epidermal growth factor (EGF) and human platelet-derived growth factor (PDGF) have been shown to promote cell growth (Chen Y, Rabinovitch PS. Platelet-derived growth factor, epidermal growth factor, and insulin-like growth factor Iregulate specific cell-cycle parameters of human diploid fibroblasts in serum-free culture. J Cell Physiol. 1989, 140 (1): 59-67, Lucarelli E, Beccheroni A, Donati D, et al. Platelet-derived growth factor sensitivity proliferation of human stromal stem cells. 2 Bioma , 24(18): 3095-100.). Both have similar effects on cell dynamics parameters, both act on the early G1 restriction point, stimulate the expression of early G1 genes such as c-fos and c-myc, and regulate the proportion of cells entering the cell cycle from a quiescent state. Gronthos S, etc. studies have confirmed that the growth of fibroblast colony-forming units (CFU-F) can only be maintained in the presence of EGF and/or PDGF under serum-free conditions, and the colony size when the two are used together is larger than that used alone ( P<0.05). Compared with colony culture containing 20% fetal bovine serum, the size of CFU-F colonies added with EGF+PDGF under serum-free conditions was significantly increased (P<0.05) (Gronthos S, Simmons PJ The growth factor requirements of STRO-1-Positive human bone marrow stromal precursors under serum-deprived conditions in vitro. Blood, 1995 85(4): 929-940). Studies on basic fibroblast growth factor (bFGF) also confirmed that it plays an important role in maintaining the proliferation and differentiation potential of mesenchymal stem cells (Bianchi G, Banfi A, Mastrogiacomo M et al. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp. CellRes. 2003, 287(1): 98-105).

In addition to cytokines, Gronthos S et al. also found that under serum-free conditions, dexamethasone and ascorbic acid are one of the key factors for the growth of CFU-F, but if only these two substances are contained without adding exogenous growth factors , cannot promote the growth of CFU-F. Therefore, adding these substances while reducing the serum concentration and adding cytokines can help the growth of pluripotent adult progenitor cells in cord blood.

Studies on the isolation and culture of bone marrow MSCs by adherent subculture have shown that the type of medium used, the initial inoculation density, and the time of initial liquid exchange are all important factors affecting the separation effect (Sotiropoulou PA, Perez SA, Salagianni M, et al.Characterization of the optimal culture conditions for clinical scale production of human mesenchymal stem cells. Stem Cells. 2006, 24(2): 462-71.). If the time for initial medium change is too long, prolonged contact with hematopoietic cells will inhibit the attached pluripotent adult stem cells from entering a proliferative state, affecting the number of cells obtained and the ability to form colonies. This may be due to environmental deterioration caused by nutrient consumption, cell death, etc., or it may be related to the promotion of cell differentiation by cytokines secreted by hematopoietic cells. However, if the time for the initial medium change is too short, the cells will not adhere sufficiently, which will also affect the final number of cells obtained. Seeding density also affects the complex interactions between cells. When primary cultured cells are exposed to the in vitro environment for the first time, the interaction between cells is very important. Cells may produce some growth-promoting active substances to promote each other's survival and growth. In addition, when the cell density is too high, there is not enough space for the adherent cells to stretch; when the cell density is too low, the time for the cells to form clones and reach the density required for passage is very long, causing the primary cells to "age" and make them grow after passage. Both the expansion ability and differentiation potential were significantly decreased.

In the previous pre-experiment, we also explored the relevant influencing factors for the isolation of umbilical cord blood multipotent adult progenitor cells under low serum conditions. Hours, using DMEM/F12 medium, the initial inoculation density of 1×10 ⁶ /cm ² group is conducive to the growth of cord blood multipotential adult progenitor cells. Although studies on bone marrow-derived MSCs have shown that low-density (1×10 ³ /cm ² ) or very low-density (1 or 3 cells/cm ² ) inoculation is favorable for MSC cell proliferation at 10-20% serum concentration. Separation and amplification (Sotiropoulou PA, PerezSA, Salagianni M, et al.Characterization of the optimal cultureconditions for clinical scale production of human mesenchymal stemcells.Stem Cells.2006,24(2):462-71.Colter DC, Class.R, DiGirolamo CM et al. Rapid expansion of recycling stem cells incultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA, 2000; 97(7): 3213-3218.). However, in the preliminary experiment, we found that under the condition of reducing the serum concentration, too low initial inoculation density was not conducive to the isolation of multipotent non-hematopoietic adult stem cells from cord blood, which was similar to the research of Tamama et al., who found that bone marrow MSCs in high Lower density cultures are more tolerant to low serum environments when cultured at a higher density (Tamama K, Fan VH, Griffith LG, et al. Epidermal growth factor as a candidate for ex vivoexpansion of bone marrow-derived mesenchymal stem cells, Stem Cells , 2006, 24(3):686-95.). At the same time, we found that the separation success rate of umbilical cord blood samples with the separation time ≤ 4 hours and the number of MNC cells ≥ 1.2×10 ⁸ was higher. Therefore, in the method of the present invention we employ these above-mentioned preferred technical factors.

The method of the present invention comprises optimizing the selection of umbilical cord blood samples, using lymphocyte separation fluid to separate umbilical cord blood mononuclear cells, culturing them in a medium containing 8-15% fetal bovine serum for 3 days, discarding unattached cells, and then replacing Containing 2-5% fetal bovine serum, human basic fibroblast growth factor (bFGF), human epidermal growth factor (EGF), human platelet-derived growth factor (PDGF-BB), MCDB-201, ascorbic acid, dextrose The adherent culture was continued in the medium supplemented with metasone and ITS, and the umbilical cord blood multipotent adult progenitor cells were purified and isolated.

Technical scheme of the present invention:

1. Human umbilical cord blood is diluted with 0.01M phosphate buffered saline (PBS) pH7.4, and mononuclear cells are separated by lymphocyte separation medium;

The umbilical cord blood is defined as: blood collected from the umbilical vein connected to the human fetal placenta. It is preferred to use human umbilical cord blood with a mononuclear cell count ≥ 1.2×10 ⁸ within 4 hours after delivery. The density of the lymphocyte separation liquid used in the cell separation of the present invention is 1.077g/cm ³ .

2. Suspend the isolated umbilical cord blood mononuclear cells in DMEM/F12 medium containing 10% fetal bovine serum, inoculate at a density of 0.5-2×10 ⁶ /cm ² , preferably 1×10 ⁶ /cm ² culture flasks (preferably pre-coated with fibronectin); culture in an incubator at 37°C, 5% CO ₂ , and 100% humidity.

3. After culturing for 48-72 hours (preferably 72 hours), discard the non-adherent cells, and replace the cell culture medium with human basic fibroblast growth factor (bFGF), human epidermal growth factor (EGF), human platelet-derived Growth factor (PDGF-BB) (the concentration of recombinant human cell growth factor added above is usually 1-10ng/ml, preferably 10ng/ml), 40% MCDB-201, 2% fetal bovine serum, 1× insulin-transfected The ferritin-sodium selenite (ITS), 10 ^-4 M ascorbic acid, 10 ^-8 M dexamethasone DMEM/F12 medium was cultured continuously, and the medium was changed 2-3 times a week.

4. When the primary culture in the above 3 is close to 70-80% confluent, the culture flask is treated with 0.25% trypsin, and then the cells are recovered, and the obtained cells are counted.

5. Inoculate the obtained cells into the medium described in the above 3 at a density of 5-8×10 ³ /cm ² (preferably 8×10 ³ /cm ² ) for culture, when the cells are close to 70-80% confluent carry out subculture;

6. Repeat the operation of 5 to carry out subculture;

The beneficial effects of the present invention are:

The invention provides a simple and feasible method for isolating multipotent adult progenitor cells from umbilical cord blood, that is, adopting the method of adding cell growth additives for adherent subculture and separation under low serum conditions. Using this method, multipotent adult progenitor cells can be effectively isolated from the middle of umbilical cord blood. The isolated cells can be repeatedly passaged in vitro, and have the same immune phenotype as the reported non-hematopoietic adult stem cells of umbilical cord blood. Cell cycle detection shows that more than 97% are in G0 /G1 phase, and has the potential of multilineage differentiation under appropriate induction conditions. When using the preferred umbilical cord blood standard and separation technology proposed by the present invention, multipotent adult progenitor cells can be isolated from 70% of the specimens, which has an obvious advantage in the success rate of separation compared with other methods. At the same time, the application of low-serum culture conditions helps to reduce the influence of serum, which is beneficial to the standardized application of further experiments and production in the future.

Detailed ways

A method for isolating multipotent adult progenitor cells from umbilical cord blood of the present invention comprises the following steps in turn:

Step 1: Dilute the umbilical cord blood in vitro with phosphate buffered saline to separate mononuclear cells with lymphocyte separation medium;

Step 2: Culture the isolated umbilical cord blood mononuclear cells in a medium containing 8-15% fetal bovine serum for 48-72 hours, and discard unattached cells;

Step 3: Replace with 2-5% fetal bovine serum, human basic fibroblast growth factor bFGF, human epidermal growth factor EGF, human platelet-derived growth factor PDGF-BB, MCDB-201, ascorbic acid, dexamethasone and 1 × insulin-transferrin-sodium selenite ITS additive medium to continue the adherent culture;

The fourth step: subculture, purification and isolation of multipotent adult progenitor cells from umbilical cord blood.

The extracorporeal umbilical cord blood is the blood collected from the umbilical vein connected to the placenta of the human fetus, preferably the human umbilical cord blood with the number of mononuclear cells ≥ 1.2×10 ⁸ within 4 hours after delivery.

Below in conjunction with specific embodiment the present invention is described in further detail:

Example 1. Isolation, purification and expansion of umbilical cord blood multipotent adult progenitor cells

After the umbilical cord blood was subjected to density gradient centrifugation in lymphocyte separation medium, mononuclear cells were collected, washed twice with DMEM/F12 medium, and the cells were suspended in DMEM/F12 medium.

The isolated mononuclear cells were inoculated into DMEM/F12 medium containing 10% fetal bovine serum at a seeding density of 1×10 ⁶ /cm ² , with 7 ml of medium in each T-25 culture bottle. Place in an incubator at 37°C, 5% CO ₂ , and 100% humidity, and cultivate for 72 hours. Discard non-adherent cells, replace the cell culture medium with 40% MCDB-201, 2% fetal bovine serum, 10ng/ml bFGF, 10ng/ml EGF, 10ng/ml PDGF-BB, 10 ^-4 M ascorbic acid, 10 ^{- 8} M dexamethasone, 1 × ITS DMEM/F12 culture medium. Change the medium 2-3 times a week. Adherent cells were seen in 70% of the cord blood samples after culture, scattered at first, and gradually formed cell clones in 12-14 days, growing in a uniform long spindle-shaped swirl. When the cells were nearly 70% confluent, the culture flask was treated with 0.25% trypsin, and then the cells were recovered, and the obtained cells were counted. An average of 7.5×10 ⁵ pluripotent adult progenitor cells were obtained from each bottle of monolayer confluent cells after digestion. The obtained cells were inoculated into the above medium at a density of 8×10 ³ /cm ² for culture, and subculture was performed when the cells were close to 70-80% confluent.

Example 2. Surface antigen characteristics and cell cycle detection of umbilical cord blood multipotent adult progenitor cells

The umbilical cord blood multipotent adult progenitor cells expanded for three generations were taken, and the culture medium was removed. The cells were washed twice with PBS, treated with trypsin (0.25%), and the cells were recovered and counted. Make 5×10 ⁵ /ml single cell suspension with PBS, add 500 μl to each tube, add 10 μl anti-human CD3, CD34, CD11a, CD29, CD105, CD45, CD44, CD73, CD49d, CD49e, CD31, CD62L , CD14, HLA-DR, CD144, vWF and anti-mouse IgG ₁ isotype control fluorescent antibody. Incubate at 4°C in the dark for 30 minutes, add 1ml PBS to wash, discard the supernatant, add 300μl PBS containing 1% paraformaldehyde, and perform flow cytometric detection. CD29, CD44, CD105, CD49d, and CD73 were positively expressed, and CD34, CD45, CD11a, CD14, CD31, HLA-DR, CD144, vWF, and CD62L were negatively expressed.

In the logarithmic phase of cell growth, digest the cells, fix with 70% cold ethanol at 4°C for 30 minutes, wash the cells twice with PBS, suspend the cells in 0.5ml PBS, add 10μl of 10mg/ml RNaseA, and incubate in a water bath at 37°C for 30min. Immediately placed in an ice bath, added 5 μg/ml PI for staining (4°C, 30 minutes), and detected by flow cytometry, the results showed that more than 97% of the cells were in the G ₀ /G ₁ phase.

Example 3. Induced differentiation characteristics of umbilical cord blood multipotent adult progenitor cells

The third-generation multipotent adult progenitor cells expanded in vitro were digested and seeded at a density of 5×10 ³ /cm ² in a 6-well plate pre-placed with a cover glass. When the cells reach 60%-70% confluence, replace each well with DMEM/F-12 containing 10 ^-7 mol/L dexamethasone, 10 mmol/L β-glycerophosphate, 0.05 mol/L vitamin C and 10% FCS 2ml osteogenic differentiation induction medium was used for induction and culture for 3 weeks, and the medium was changed twice a week. On the 14th and 21st day of induction, the slides were taken out, fixed, and observed for morphological changes and histological staining.

The third-generation multipotent adult progenitor cells expanded in vitro were digested and seeded at a density of 5×10 ³ /cm ² in a 6-well plate pre-placed with a cover glass. When the cells reached ⁶⁰ %-70% confluence, each well was replaced with DMEM/F- 12 Adipogenic differentiation induction medium 2ml was used for induction culture, and the medium was changed once every 3-4 days. When the formation of fat vacuoles in the cytoplasm was observed, the culture medium was aspirated, washed once with PBS, fixed with 70% alcohol for 10 minutes, and washed with Oil Red dye solution staining.

After 14 days of umbilical cord blood multipotent adult progenitor cells in an osteogenic environment, Alizarin red staining showed red calcified matrix deposits; Von Kossa staining showed black calcified matrix deposits. Under the adipogenic induction environment, the Oil Red stain stained positively. It indicated that the cells had the potential to differentiate into osteoblasts and adipocytes.

Claims (10)

1. A method for isolating multipotent adult progenitor cells from umbilical cord blood, comprising the following steps in turn: Step 1: Dilute the umbilical cord blood in vitro with phosphate buffered saline to separate mononuclear cells with lymphocyte separation medium; Step 2: Culture the isolated umbilical cord blood mononuclear cells in a medium containing 8-15% fetal bovine serum for 48-72 hours, and discard unattached cells; Step 3: Replace with 2-5% fetal bovine serum, human basic fibroblast growth factor bFGF, human epidermal growth factor EGF, human platelet-derived growth factor PDGF-BB, MCDB-201, ascorbic acid, dexamethasone and 1 × insulin-transferrin-sodium selenite ITS additive medium to continue the adherent culture; The fourth step: subculture, purification and isolation of multipotent adult progenitor cells from umbilical cord blood. 2. The method for isolating multipotent adult progenitor cells from umbilical cord blood according to claim 1, characterized in that the in vitro umbilical cord blood is collected from the umbilical vein connected to the human fetal placenta, preferably 4 hours after delivery. Within, the human umbilical cord blood with the number of mononuclear cells ≥ 1.2×10 ⁸ . 3. The method for isolating multipotent adult progenitor cells from umbilical cord blood according to claim 1, characterized in that the second step is: suspending the isolated umbilical cord blood mononuclear cells in a medium containing 10% fetal bovine serum DMEM/F12 medium was inoculated in a culture bottle at a density of (0.5-2)×10 ⁶ /cm ² ; placed in an incubator at 37° C., 5% CO ₂ , and 100% humidity for 72 hours. 4. The method for isolating multipotent adult progenitor cells from umbilical cord blood according to claim 3, characterized in that the culture flask is pre-coated with fibronectin. 5. The method for isolating multipotent adult progenitor cells from umbilical cord blood according to claim 3, characterized in that the density is preferably 1×10 ⁶ /cm ² . 6. The method for isolating multipotent adult progenitor cells from umbilical cord blood according to claim 1, characterized in that the cell culture medium in the third step contains 40% MCDB-201, 2-5% fetal bovine serum , 1-10ng/ml human basic fibroblast growth factor bFGF, 1-10ng/ml human epidermal growth factor EGF, 1-10ng/ml human platelet-derived growth factor PDGF-BB, 1× insulin-transferrin- Sodium selenite ITS, 10 ^-4 M ascorbic acid, 10 ^-8 M dexamethasone in DMEM/F12 medium. 7. The method for isolating multipotent adult progenitor cells from umbilical cord blood according to claim 1, characterized in that in the third step, the medium is changed 2-3 times a week. 8. The method for isolating multipotent adult progenitor cells from umbilical cord blood according to claim 6, characterized in that the fourth step comprises: a. When the primary culture in the third step is close to 70-80% confluence, the culture flask is treated with trypsin, and then the cells are recovered, and the obtained cells are counted; b. Inoculate the cells obtained in a. at a density of (5-8)×10 ³ /cm ² into the medium described in the third step above for culture, and subculture when the cells are close to 70-80% confluence; repeat Carry out the operation of b and carry out subculture. 9. The method for isolating multipotent adult progenitor cells from umbilical cord blood according to claim 1, characterized in that the concentration of the phosphate buffer is 0.01M, pH=7.4; the specific gravity of the lymphocyte separation solution is 1.077g/cm ³ . 10. The method for isolating multipotent adult progenitor cells from umbilical cord blood according to claim 1, characterized in that the preferred concentration of fetal bovine serum in the second step is 10%, and the preferred concentration of fetal bovine serum in the third step is 2%.