WO2009030092A9

WO2009030092A9 - Culture medium and method for in vitro culturing human adult primary mesenchymal stem cells on a large scale, primary mesenchymal stem cells obtained by the method, the uses thereof

Info

Publication number: WO2009030092A9
Application number: PCT/CN2007/070637
Authority: WO
Inventors: 赵春华; 韩钦; 李静; 孙昭; 胡建立; 朱雅姝; 卢姗; 边春景
Original assignee: 中国医学科学院基础医学研究所
Priority date: 2007-09-05
Filing date: 2007-09-05
Publication date: 2010-04-22
Also published as: CN101821383B; WO2009030092A1; CN101821383A

Abstract

A method for in vitro preparing human adult primary mesenchymal stem cells (pMSCs, which are positive for Flk1 marker and Nanog marker) derived from human adult tissues (marrow) on a large scale and the products obtained by the same are provided. Uses of the method and the products obtained by the same in inhibiting the proliferation of tumor cells are also provided. By modifying the key processes and parameters of the preparation of pMSCs, for example simplifying the process of magnetic cell sorting, using human serum culture medium instead of bovine serum culture medium, and using the culture condition at low concentration oxygen (2%), the aim of preparing pMSCs on a large scale is achieved. By using the property of expressing Nanog gene of pMSCs, the novel use of pMSCs in inhibiting the proliferation of tumor cells is invented.

Description

Culture medium, method and obtained original mesenchymal stem cell for human adult primitive mesenchymal stem cells in vitro and its application

The present invention relates to a method for producing human mesenchymal stem cells (pMSCs, cell markers labeled Flkl, Nanog positive) derived from human adult tissue (bone marrow) in vitro and in large scale production thereof, and a preparation product thereof, and the present application also relates to the use of the cells for inhibiting tumor cells Proliferation application. Background technique

Stem cells are a primitive cell population with self-renewal ability and multilineage differentiation potential in vivo. All tissues and organs of the human body are differentiated from stem cells. Stem cells can be roughly divided into two types, one is embryonic stem cells and the other is adult stem cells. Embryonic stem cells are the most primitive stem cells, which can be infinitely replicated and differentiated into any tissue cells. However, due to the current scientific and technological level and ethical restrictions, it is difficult to apply to practical applications in the short term. Recent studies on adult stem cells have shown good prospects for future life sciences.

At present, the general method for culturing and purifying adult stem cells (mesenchymal stem cells) mainly uses magnetic beads to sort mesenchymal stem cells, and is cultured under the condition of conventional oxygen content (20% O ₂ ), and the medium used is usually added to the animal source. Serum, such as bovine or horse serum. On the one hand, mesenchymal stem cells cultured by conventional methods are difficult to meet the requirements of practical applications in terms of stem cell biological characteristics and stem cell differentiation potential; on the other hand, these conventional methods have many disadvantages, for example, when conventional When a medium containing 100-200 ml/L of animal serum is used, it usually has the following disadvantages: First, the average doubling time of the cultured mesenchymal stem cells is long, and the growth rate is not uniform, and cannot be prepared within a prescribed time. The number of cells required; Secondly, the animal serum used inevitably has risk factors such as xenogeneic immune rejection and animal pathogenic infection. In addition, the commonly used magnetic bead sorting process also has a number of disadvantages. First, the cost of use is relatively high. The magnetic beads currently used for cell separation are more expensive. About 10 ⁸ cells per bone marrow sample (20-30ml) need to be separated. The cost of preparation for a single human pMSCs will increase by at least 4,000 yuan. Secondly, the magnetic bead sorting process has a longer operation time in vitro, which greatly increases the chance of cell contamination and has a great influence on the activity of stem cells. Third, the selection of magnetic beads is either positive or negative. When the separation method is selected, different numbers of cells will be lost; the magnetic beads bound to the cells cannot be completely removed in the subsequent process, and finally can be transplanted into the human body with the stem cells, and the magnetic beads have the possibility of negatively affecting human health. . For the culture conditions of conventional oxygen content (20% O ₂ ), since the oxygen concentration in the body is significantly lower than this value, the average oxygen concentration of the arterial blood is about 12%, the average oxygen concentration of the tissue is about 3%, and the main accumulation site of pMSCs. The oxygen concentration of a bone marrow is 1%-7%, and the concentration of oxygen in the embryo body enriched in stem cells is lower. Excessive concentrations of oxygen cause cells to produce large amounts of free radicals, which have a damaging effect.

Primitive mesenchymal stem cells (pMSCs) are derived from human adult tissues and are present in various tissues of human body. They have similar self-renewal and multilineage differentiation potential of embryonic stem cells, and can be induced to differentiate into various tissue cells under specific circumstances, and cells It has low immunogenicity, can form a stable chimera in the recipient after transplantation, induces specific immune tolerance in the body, and has the characteristics of allogeneic application. The prepared pMSCs have various biological functions in vivo and in vitro, including rebuilding blood, regenerating blood vessels, reducing fibrotic damage, inhibiting immune rejection, etc., and can be used for multi-tissue organs such as leukemia, myocardial infarction, progressive liver injury, diabetes, and the like. Therapeutic applications of traumatic diseases. Summary of the invention

There is still a need to overcome the deficiencies of the above-described methods and to rapidly provide adult stem cells that meet the needs of the actual application, and the applicant has provided the present application to meet the above needs.

The present application provides a medium for culturing raw mesenchymal stem cells pMSCs derived from human adult tissues, including cell basal medium and human serum, the final concentration of said human serum being 10-100 ml/L.

Further, the culture medium provided herein preferably has a final concentration of human serum of 10-30 ml/L. A more preferred human serum has a final concentration of 20 ml/L. The culture medium described in the present application preferably has human serum as human peripheral or cord blood serum.

In another aspect, the medium provided by the present application further comprises human serum albumin, epidermal growth factor EGF and platelet-derived growth factor PDGF, wherein the final concentration of the human serum albumin is 5-20 mg/ml, and the epidermal growth The final concentration of the factor is 1-100 ng/ml, and the final concentration of the platelet-derived growth factor is 1-100 ng/ml. A preferred final concentration of human serum albumin is 5-10 mg/ml, a preferred final concentration of epidermal growth factor is 10-30 ng/ml, and a preferred final concentration of platelet-derived growth factor is 10-30 ng/ml. More preferred human albumin The final concentration is 10 mg/ml, and the more preferred final concentration of epidermal growth factor is 10 ng/ml, and the more preferred final concentration of platelet-derived growth factor is 10 ng/ml.

Further, the medium provided by the present application is used to culture bone marrow-derived primitive mesenchymal stem cells.

In still another aspect, the present application provides a method of culturing original mesenchymal stem cell pMSCs derived from human adult tissue in vitro, the method comprising the following steps:

1) isolating human adult tissue mononuclear cells and suspending in the medium as a primary cell suspension;

2) The primary cell suspension is inoculated at a seeding density of ¹ ×10 ⁶ cells/ml in a plastic petri dish containing the medium; in a volume ratio, at 2% (1-5%, preferably 2%) The oxygen concentration, 5% C0 ₂ concentration and 37 ° C, cultured in a saturated humidity incubator; repeated adherent culture 3-4 times to obtain human mesenchymal stem cells of adult tissues.

Further, the method is for culturing primitive mesenchymal stem cells derived from bone marrow. In another aspect, the application provides the original mesenchymal stem cells obtained by the above method. Further, the original mesenchymal stem cells have an immunophenotype of Flkl-positive and express the Nanog gene.

In a further aspect, the application provides the use of the primordial mesenchymal stem cells for the preparation of a medicament for inhibiting tumor cell proliferation.

In another aspect, the application provides a pharmaceutical composition comprising the original mesenchymal stem cells.

In a further aspect, the present application provides an adult original mesenchymal stem cell culture PEPSC, which has the accession number CGMCC No. 2152, and was deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on September 4, 2007, including the original Mesenchymal stem cells.

In another aspect, the application provides the adult primordial mesenchymal stem cell culture PEPSC, wherein the primordial mesenchymal stem cells have an immunophenotype of FM positive and a Nanog gene.

In a further aspect, the application provides the use of the adult primordial mesenchymal stem cell culture PEPSC for the preparation of a medicament for inhibiting tumor cell proliferation.

In another aspect, the application provides a pharmaceutical composition comprising the adult original mesenchymal stem cell culture PEPSC. BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1: Comparison of phenotypic characteristics of pMSCs cultured under conventional oxygen concentration and hypoxic concentration. A low oxygen concentration pMSCs, B conventional oxygen concentration pMSCs.

Figure 2: Comparison of cell cycle assays of pMSCs cultured under conventional oxygen concentration and hypoxic concentration. A low oxygen concentration pMSCs, B conventional oxygen concentration pMSCs.

Figure 3-1: Study on the differentiation ability of pMSCs cultured under conventional oxygen concentration and hypoxia concentration I: Two weeks after induction into osteoblasts, Von Kossa staining was used to detect calcium deposition. A low oxygen concentration pMSCs, B conventional oxygen concentration pMSCs.

Figure 3-2: Study on the differentiation ability of pMSCs cultured under conventional oxygen concentration and hypoxic concentration Π: After 2 weeks of induction to fat cells, oil red O staining. A low oxygen concentration pMSC, B normal oxygen concentration pMSC.

Figure 3-3: Study on the differentiation ability of pMSCs cultured under conventional oxygen concentration and hypoxic concentration III: Induction of differentiation into the endothelium on Matrigel gel After 48 hours, a grid-like tube-like structure was formed. A low oxygen concentration pMSCs, B conventional oxygen concentration pMSCs.

Figure 4: Comparison of proliferative capacity of pMSCs cultured under conventional oxygen concentration and hypoxic concentration. Figure 5: Comparison of cell morphology in different media of pMSCs:

A: morphology of pMSCs in 20 ml/L fetal bovine serum medium; B: morphology of pMSCs in 20 ml/L peripheral blood serum system; C: morphology of pMSCs in 20 ml/L cord blood serum medium.

Figure 6: Phenotypic test results of pMSCs in different media:

A. Immunophenotypic results of pMSCs in fetal bovine serum medium; B. Immunophenotypic results of pMSCs in peripheral blood serum medium; C. Immunophenotypic results of pMSCs in umbilical cord serum medium.

Figure 7: Comparison of cell cycle of pMSCs in different media.

Figure 8-1: Study on the multi-directional differentiation ability of pMSCs in different media I, (Results of oil red "O" staining after three weeks of induction of adipogenic induction system):

A: fetal bovine serum medium; B: peripheral blood serum medium; C: umbilical cord serum medium,

P3: 3rd generation; P7: 7th generation.

Figure 8-2: Study of the multi-directional differentiation ability of pMSCs in different media II, (Von Kossa staining results after three weeks of induction of osteogenic induction system: brown shows calcium matrix deposition): A: fetal bovine serum medium; B: peripheral blood serum medium; C: umbilical cord serum medium,

P3: 3rd generation; P7: 7th generation.

Figure 8-3: Study on the multi-directional differentiation ability of pMSCs in different media III, (immunofluorescence staining results after three weeks of neurogenic induction):

P3: 3rd generation; P7: 7th generation.

Figure 9: Growth curves of pMSCs in three different media:

1: fetal bovine serum medium; 2: peripheral blood serum medium; 3: umbilical cord serum medium, the data of the three groups of media were not statistically significant.

Figure 10: RT-PCR detection of human pMSCs expressing Nanog, Dkk-1 results.

Figure 11-1: Tumor cell proliferation assay after co-culture with pMSCs:

K562: K562 cells cultured alone; K562+pMSC: K562 co-cultured with pMSCs; MCF7: MCF7 cells cultured alone; MCF7+pMSC: MCF7 co-cultured with pMSCs; HL60:: HL60 cells cultured alone; HL60+pMSC: HL60 Co-culture with pMSCs.

These results have been analyzed for the mean standard deviation (+ S.D.) calculated after six independent experiments.

Figure 11-2: Detection of proliferation of K562 by the addition of neutralizing antibodies or transwell when K562 is co-cultured with pMSCs:

Group 1: K562 cells cultured alone; Group 2: K562 cells co-cultured with pMSCs; Group 3: K562 cells co-cultured with pMSCs, anti-Dkk-1 added to the medium; Group 4: Co-cultured with pMSCs K562 cells, and use transwell to separate the two cells.

Figure 11-3: Interference effects of Dkk-1 and Nanog after transfection of RNA interference vectors with pMSCs

A: Quantitative PCR detection of Nanog expression: SiM: expression of Nanog in pMSCs transfected with empty vector control; SiN: expression of Nanog in pMSCs transfected with SiNanog vector

B: Quantitative PCR detection of Dkk-1 expression: SiM: expression level of Dkk-1 in pMSCs transfected with empty vector control; SiD: Table of Dkk-1 in pMSCs transfected with SiDKKl vector Yield; SiN: expression level of Dkk-1 in pMSCs transfected with SiNanog vector;

C: Western blot was used to detect the expression of Nanog and Dkk-1.

Figure 11-4: pMSCs after interference with Nanog and Dkk-1 were co-cultured with K562 to detect changes in K562 cell cycle:

Group 1: K562 cells cultured alone; Group 2: K562 cells co-cultured with pMSCs transfected with empty vector control; Group 3: K562 cells co-cultured with pMSCs transfected with SiDKKl vector; Group 4: Transfected with SiNanog Vectors of pMSCs co-cultured with K562 cells.

Figure ll-5: pMSCs after interference with Nanog and Dkk-1 were co-cultured with K562, and the accumulation of β-catenin in K562 cells was detected by Western blot.

Figure 11-6: pMSCs after interference with Nanog and Dkk-1 were co-cultured with K562, and the gene expression of cell cycle-associated proteins in K562 was detected by quantitative PCR:

A: Quantitative PCR was used to detect P21 expression; B: Quantitative PCR was used to detect P27 expression; C: Quantitative PCR was used to detect c-myc expression; D: Quantitative PCR was used to detect cyclinD2 expression.

K562: K562 cells cultured alone; K562+SiM: K562 cells co-cultured with pMSCs transfected with empty vector control; K562+SiD: K562 cells co-cultured with pMSCs transfected with SiDKKl vector; K562+SiN: with K562 cells co-cultured with pMSCs stained with SiNanog vector.

The present application will be described in detail below with reference to the accompanying drawings and embodiments, wherein the description and the embodiments of the present application are merely illustrative and not limiting, and those skilled in the art can fully combine the existing The technology makes various improvements and modifications to the present application. However, the improvements and modifications made will not go beyond the spirit of the present application and fall within the scope of the claims as claimed. detailed description

SUMMARY OF THE INVENTION The object of the present application is to provide an improved method for the isolation and culture of human adult tissue (bone marrow) derived pMSCs in vitro.

The method for isolating and culturing human pMSCs provided by the present application is to obtain a certain amount of bone marrow tissue. After the pretreatment process, the primary seed cells are collected, and the collected cells are seeded in a plastic culture dish and added to a specific cell culture medium. Culture; Purify cells by adherent culture method, purify cells by using the unique adherence characteristics of cultured cells, and multiply cells Sub-adherent, de-suspended, to achieve the purpose of purifying pMSCs and removing miscellaneous cells. The biggest advantage of this method compared with the traditional stem cell sorting method is that the magnetic bead sorting purification process is abandoned. The magnetic bead sorting process is costly to use. The magnetic beads currently used for cell separation are more expensive. About 10 ⁸ cells per bone marrow sample (20-30ml) need to be separated, and the cost of preparation of single human pMSCs will increase at least. 4,000 yuan; Secondly, the magnetic bead sorting process has a longer operation time in vitro, which greatly increases the chance of cell contamination and has a great influence on stem cell activity. Third, the separation of magnetic beads is performed by positive or negative selection. Different numbers of cells will be lost; the magnetic beads bound to the cells cannot be completely removed in the subsequent process, and finally can be transplanted into the human body with stem cells, and these magnetic beads have the possibility of negatively affecting human health. By adopting the new method of the present application, it is possible to amplify a sufficient quantity and quality (scale production) of stem cells within a prescribed time and at a lower cost, and to achieve a purity of more than 85%, which meets the quality standard of the finished product, thereby Meet the needs of therapeutic applications. The method provided by the present application greatly simplifies the cell purification process step, improves the production efficiency, and reduces the production process cost, and the usual stem cell culture method can only be prepared in a small amount, and is only suitable for scientific research, and the proliferation ability and culture of the cell itself. The cost is difficult to meet the requirements of large-scale production preparation.

The pMSCs culture preparation method provided by the present application is different from the conventional stem cell culture in that a culture condition of a low concentration of 0 ₂ is employed in the whole process of preparation and culture of pMSCs. In vitro cell culture is generally carried out at a concentration of 20% O ₂ , and the oxygen concentration in the body is significantly lower than this value. The average oxygen concentration of arterial blood is about 12%, the average oxygen concentration of tissue is about 3%, and the main accumulation site of pMSCs. The oxygen concentration of a bone marrow is 1%-7%, and the concentration of oxygen in the embryo body enriched in stem cells is lower. Usually, the so-called low oxygen concentration is more in line with the physiological state. Excessive concentration of oxygen causes the cells to produce a large amount of free radicals, and instead has a stress-induced effect. In the present application, pMSCs were cultured under physiological conditions (2% volume concentration of 0 ₂ ), which is more suitable for growth, proliferation and maintenance of undifferentiated state of pMSCs than conventional oxygen concentration conditions (20% volume concentration of 0 ₂ ). The hypoxic concentration culture environment used in the present application is provided by a constant temperature three-gas incubator, and has simple operation and good repeatability, and is suitable for large-scale cultivation.

In addition, the present application provides a new medium that is different from conventional bovine serum medium, which contains human serum. At present, the conventional adult stem cell culture (base) system uses fetal bovine serum culture medium, and the fetal bovine serum added to the system is heterologously derived from the human species, and there is a potential risk of contaminating the exogenous xenogeneic pathogen. Similar to serum one The stem cell culture of the class must be added to the humanization transformation will be the main improvement direction of the future development of stem cell production technology. The present application first invented a human serum medium instead of fetal bovine serum medium, and successfully prepared adult stem cells of the same quality as the original medium under the system (Table 1).

Further, the medium provided by the present application also includes human albumin products. The serum in the preferred medium of the present application is derived from human peripheral blood serum/umbilical cord blood serum. Human serum and human serum albumin replace the fetal bovine serum and bovine serum albumin components in the conventional medium formulation, respectively, thereby realizing the humanization of all heterogeneously added components in the culture solution. At the same time, another significant advantage is: the human umbilical cord blood serum used in the medium comes from the cord blood storage (library) mechanism, and the cord blood serum is a by-product discarded after the cord blood hematopoietic stem cells are collected before the cord blood is stored. However, cord blood serum contains a large number of cytokines and nutrients, which can be used as a substitute for fetal bovine serum, and the source of cord blood and its collection and processing techniques fully ensure that the serum itself is clean and non-polluting, and its source is very convenient (umbilical cord The recycling of blood processing wastes ensures the supply of medium supplements during the large-scale preparation of adult stem cells, and also greatly reduces the cost of preparation of adult stem cells (the serum products are expensive and rely on imports).

The adult stem cell pMSCs were prepared in vitro using the above human serum medium. The results showed that the quantity and quality of the pMSCs prepared according to the method of the present application reached the quality standard and fully met the requirements for clinical treatment (Fig. 5 - Fig. 9). . Moreover, the use of this medium avoids the possibility of heterogeneous immune rejection and the risk of unknown virus infection in animal serum, and the clinical application is safer and more reliable.

The peripheral blood serum and cord blood serum used in the present application are all derived from the process products complying with the GMP requirements, and the human serum albumin is a listed product in conformity with the national drug standard, and the above conditions ensure the process technology using the human serum medium. The smooth progress of adult stem cell industrialization.

In the above method, the primary cells are repeatedly cultured 3-4 times in a dedicated medium to obtain human primordial mesenchymal stem cells which meet the purity requirements.

In the above method, the harvested cells are seeded in a plastic petri dish at a seeding density of 5χ 10 ⁴ cells/ml;

In the above method, the culture conditions of the culture are 5% C〇 ₂ , 2% 0 ₂ , 37 ° C and a saturated humidity incubator.

The isolated culture method of the present application is suitable for isolating pMSCs from bone marrow tissues, cells The purification method is applicable to pMSCs of various tissue sources. The separation and culture method is simple, efficient, and low in cost. The cultured pMSCs have CD31-, CD34\CD45- and HLA-DR- (negative), and CD29 ⁺ , CD44 ⁺ , CD105+ and FM ⁺ (positive) phenotypes.

Human pMSCs prepared by the method of the present invention can express specific cell markers Flkl and Nanogo Flkl are specific cell surface markers for controlling the purity and cell quality of pMSCs in large scale preparation; Nanog is a transcription factor involved in DKK-1 gene. The transcriptional regulation, in turn, affects the expression of its protein, and regulates the accumulation of β-catenin in the cell through the Wnt signaling pathway, affecting the β-catenin nuclear-promoting cells, and inhibiting the proliferation of tumor cells from the G1 phase to the S phase. Through this principle, the expression of Nanocell-derived pMSCs can inhibit the proliferation of tumor cells. The mechanism of inhibition of tumors is through the secretion of the soluble factor DKK-1 molecule, which inhibits tumor cells in the G0/G1 quiescent phase.

The pMSCs obtained in the present application can be applied to inhibit tumor cell proliferation. In vitro, pMSCs were co-cultured with various human hematological tumor cells, and the Nanog gene was expressed by pMSCs to regulate Dkk protein expression, and finally the Wnt signaling pathway was used to significantly inhibit tumor cell proliferation (Fig. 11). Further research on its mechanism of action, first using neutralizing antibody technology to prove that pMSCs affect tumor cell proliferation mainly through the secretion of Dkk protein, after using RNAi technology to interfere with the expression of Nanog expression or Dkk secretion of pMSCs, pMSCs on the tumor cell cycle The impact is significantly weakened. pMSCs have the effect of inhibiting the proliferation of tumor cells, but mature stromal cells do not have this effect. This application is the first to express Nanog protein by using pMSCs (current studies suggest that Nanog protein is not expressed except for embryonic stem cell expression) The expression of Dkk protein is specifically regulated, so that the Dkk protein can influence the cycle of tumor cells through the wnt signaling pathway, and finally inhibit the growth and proliferation of tumor cells.

The pMSCs obtained by the present application and the method for inhibiting the same are applicable to hematological tumors such as erythroleukemia and promyelocytic leukemia, and provide basic theoretical and clinical research evidence for treating other system tissues such as breast cancer, and are tumors. Treatment offers a new approach.

The applicant will be described in more detail in conjunction with the following examples, which will be understood by those skilled in the art that the following examples are only used to illustrate the invention and not to limit the scope of the invention. . Unless otherwise stated, the experimental methods in the following examples are all conventional methods. Example 1: In vitro scale-up preparation culture method of human pMSCs

First, reagents and medium

The specific cell culture medium used for the large-scale preparation of human pMSCs used in the present application includes cell basal medium, human serum, human serum albumin, epidermal growth factor (EGF) and platelet-derived growth factor (PDGF); The final concentration is 10-100 ml/L, the final concentration of the human serum albumin is (5-20 mg/ml), and the final concentration of the epidermal growth factor is 1-100 ng/ml, the platelet-derived growth factor The final concentration is l-100 ng/ml.

Wherein the preferred concentration of the human serum is 10-30 ml/L, most preferably 20 ml/L _; the final concentration of the human serum albumin is preferably 5-10 mg/ml, particularly preferably 10 mg/ml; The final concentration of epidermal growth factor is preferably 10-30 ng/ml, particularly preferably 10 ng/ml _; the final concentration of the platelet-derived growth factor is preferably 10-30 ng/ml, particularly preferably 10 ng/ml.

The cell basal medium may be any one of DMEM/F 12, MCDB-201, DMEM, MEM, RPMI1640, DMEM, M199, BME, IMEM, or the like, or any combination thereof.

Among them, type I collagenase was purchased from Sigma; trypsin was purchased from Gibco; DMEM/F12 (ie DF12), M199, MEM, RPMI1640, BME, IMEM and DMEM were purchased from GIBCO; MCDB-201 was purchased from Sigma; EGF was purchased from Gibco; PDGF, VEGF and bFGF were purchased from Sigma; hydrocortisone was purchased from Sigma; FCS and horse serum (HS) were purchased from Hyclone; human albumin was purchased from Harbin Shiheng Bioengineering Company; Human prothrombin is a gift from Shanghai Lai Shi Blood Products Co., Ltd.; Human umbilical cord blood serum is donated by Beijing Cord Blood Bank; Human peripheral blood serum is self-made (natural coagulation is obtained, which is a classic textbook method). 2. In vitro large-scale preparation and culture method of human pMSCs

The method for in vitro large-scale culture of human pMSCs cells provided by the present application is as follows:

1. Obtain human adult tissue primary cell suspension

1.1 Human adult tissue mononuclear cells were obtained according to the methods disclosed in the prior art, and human adult tissue mononuclear cells were suspended in a medium containing 2% human serum to obtain a human adult tissue primary cell suspension.

1.2 Count the viable cells by placental blue staining, and adjust the cell concentration to about Ι χ ΙΟ ⁶ 2. Isolation and culture of human pMSCs

2.1 15 ml of a mononuclear cell suspension of the above density (1 x ^{10 6} /ml) was inoculated into a 175 flask of the cell culture medium of the present application. The conditions of the three-gas culture incubator were previously set to 1-5% 0 ₂ , preferably 2% 0 ₂ , 5% C0 ₂ , 37 ° C, and pre-equilibrated for 3-5 hours, and then the cells were placed in an incubator.

2.2 After 24 hours of culture, a small amount of cells adhered to the cells, and the primary pMSCs grew in a clone-like manner, and the cells were fusiform. The suspended cells are discarded, and the freshly prepared cell culture medium of the present application is added to continue cultivation in a 1-5% 0 ₂ , preferably 2% 0 ₂ incubator, and the medium is changed every 3 days during the culture.

2.3 When the cells were grown to 70%-80% confluence, the medium was aspirated, the cells were washed once with 10 ml of physiological saline, digested with 0.25% trypsin 10 ml for 2 minutes, and then 0.2 ml of human serum was added to terminate the digestion. lOOOOrpm, centrifuge at room temperature for 5 minutes, discard the supernatant, add the culture solution to resuspend the cells, count, subculture at a cell density of 5x 10 ⁴ /ml per bottle at 1-5% 0 ₂ , preferably 2% 0 ₂ low oxygen concentration In the three gas incubator. Repeat 2.3 steps 3-4 times (repeated passage of adherent culture) to obtain the desired pMSCs cells.

3. Cryopreservation of human pMSCs

3.1 The cultured expanded pMSCs were digested with 0.25% trypsin 10 ml for 2 minutes, then 0.2 ml of human serum was added to terminate the digestion, and the mixture was centrifuged at 100 rpm for 5 minutes.

3.2 Discard the supernatant, suspend the cells in an appropriate amount of DF12 medium, mix with an equal volume of cell cryopreservation solution, make the cell concentration less than lx 10 ⁷ cells/ml, and place in a cryotube.

3.3 The cells were cryopreserved in liquid nitrogen after being cooled by process control.

The cryopreserved cell culture is named adult original mesenchymal stem cell culture

PEPSC, deposited with the General Microbiology Center of the China Microbial Culture Collection Management Committee on September 4, 2007, has a deposit number of CGMCC No. 2152.

4. Resuscitation of human pMSCs

4.1 The frozen cells were removed from the liquid nitrogen and rapidly thawed in a 37 ° C water bath.

4.2 Place the cells in 20 ml of DF12 medium containing 20 ml/L human serum, 1500 rpm Centrifuge for 5 minutes.

4.3 The supernatant was discarded, the cell concentration is adjusted to a bottle lx lO ⁶ cells were subcultured at 2% 0: low oxygen concentration incubator. Example 2: Comparison of Biological Characteristics of Cultured pMSCs with Conventional Oxygen Concentration and Low Oxygen Concentration of the Present Application

The pMSCs obtained by the culture method described in the present application were compared with the pMSCs obtained under the conventional oxygen concentration conditions (the other culture conditions except the oxygen concentration were the same as in the present application), and the following biological characteristics were compared and measured.

1. Immunophenotypic identification of human pMSCs

The spindle cell phenotype obtained after culture was detected by direct immunofluorescence, and the cells were labeled with anti-human CD29, CD44, CD105, Flk-1, labeled with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). CD31, CD34, CD45 and HLA-DR antibodies (all of which were purchased from BD) were labeled for flow detection, and the flow cytometry was BD FACScan (Becton Dickinson)

The cell cycle was measured by flow cytometry. The cells were fixed with 80% cold ethanol at 4 ° C for 1 h, washed twice with PBS, resuspended in 0.5 ml PBS, added with RnaseA (100 g/ml), and incubated at 37 °C for 30 minutes. Stained with propidium iodide (Sigma, 5 μg/ml) before the measurement. Cell cycle was analyzed with ModiFit software (Becton Dickinson).

The phenotypic results showed that the pMSCs cultured in the conventional oxygen concentration and the low oxygen concentration had no obvious region, and the positive rate of CD29, CD44, CD105 and Flk-1 in the spindle cells obtained from the expanded culture was over 95%, CD31. The positive rates of CD34, CD45 and MHC class II molecules are below 5% (Fig. 1). The results of cell cycle assay showed that most of the spindle cells obtained in the expanded culture were in G0/G1 phase, while the proportion of cells in G2-M phase and S phase was very low (Fig. 2).

2. Study on the multi-directional differentiation ability of pMSCs

The pMSCs cultured in the 10th generation conventional oxygen concentration and low oxygen concentration were induced to differentiate into osteoblasts, fat and endothelium by conventional methods. It was found that there was no significant difference in the differentiation ability of the two pMSCs (Fig. 3-1, 3-2). , 3-3). 3. Proliferation ability of pMSCs

The 10th generation of pMSCs cultured at the conventional oxygen concentration and the low oxygen concentration were taken, and the cells were seeded in a 24-well plate at ⁵ ×10 ³ cells/well, then 3 wells were digested every 24 hours, and the cells were collected and 0.4% of the fetuses were used. Panlan counts live cells and then plots the growth curve. The results showed that the proliferation of pMSCs cultured at low oxygen concentration was faster, and the doubling time was about 26 hours. The doubling time of conventional oxygen concentration pMSCs was about 30 hours, and the difference was statistically significant (t test, p<0.05) (Fig. 4).

Example 3: Study comparison Results of new medium and conventional medium provided by the present application on human pMSCs cell culture

The specific cell culture medium used for the large-scale preparation of human pMSCs, including cell basal medium, human serum, human serum albumin, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF). The isolated pMSCs were separately cultured in the above medium containing different human serum concentrations, wherein the final concentrations of human serum were 10 ml/L, 20 ml/L, 50 ml/L and 100 ml/L, and the final concentration was 20 ml/L. The conventional medium of fetal bovine serum was used as a control, and the cells were cultured for 10 generations under the conventional culture conditions. The pMSCs cultured in each experimental group and the control group were detected, and the differentiation ability and growth rate of the cells cultured in the experimental group were found. The biological characteristics such as the phenotype are comparable to those of the control cultured cells, and are similar to those of the fetal bovine serum cultured mesenchymal stem cells reported in the related literature. (The results are not shown).

In addition, the applicant cultured the isolated pMSCs with different concentrations of human peripheral blood serum medium, different concentrations of human umbilical cord serum medium culture and standard conventional fetal bovine serum medium, and separately obtained the cells of the cells. The characteristics of the study were compared. The concentration of human peripheral blood serum and human umbilical cord serum was compared by differentiating the differentiation ability, growth rate and phenotype of human pMSCs at different serum concentrations. The results showed that the best concentration was obtained by using serum concentration of 20 ml/L (Table). 1). Human serum concentration differentiation ability proliferation rate phenotype (Flkl+) ratio

10ml L + + + + + +

20ml L + + + + + + + + + + + + +

50ml L + + + + + + +

lOOml L + + +

Table 1: Comparison of characteristics of human pMSCs at different serum concentrations (the higher the number + indicates the higher the index) The following results were obtained for human pMSCs cultured at a concentration of 20 ml/L human serum: 1. Morphological and immunophenotypic identification of human pMSCs And cell cycle assay

Human pMSCs were isolated as described above and placed in 20 ml/L fetal bovine serum medium, 20 ml/L human peripheral blood serum medium, and 20 ml/L human umbilical cord serum medium in three different cell culture systems. Subculture, cell culture until the third generation was observed under a microscope, the cells were fusiform, and the fish were arranged in a cluster. There was no difference in cell morphology under the three system cultures. And after passing the 7th generation, the shape remains unchanged. (Figure 5)

The spindle cell phenotype obtained after culture was detected by direct immunofluorescence. The cells were labeled with anti-human CD29, CD44, CD105, Flk-1, CD31 labeled with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). The CD34, CD45, and HLA-DR antibodies (all of which were purchased from BD) were labeled and subjected to flow cytometry. The flow cytometer was BD FACScan (Becton Dickinson). The phenotypic test results showed that there was no significant difference between 20 ml/L fetal bovine serum medium, 20 ml/L peripheral blood serum medium and pMSCs cultured in 20 ml/L umbilical cord serum medium. The phenotypic identification of the spindle cells obtained from the third generation was performed. The positive rates of CD29, CD44, CD105, MHC-1 and Flk-1 were over 95%, CD31, CD33, CD34, CD45, CD117 and MHC II. The positive rate of the class of molecules is below 5%. The phenotypic identification of spindle cells passaged to passage 7 showed that the positive rates of CD29, CD44, MHC-I, CD105 and Flk-1 were over 95%, CD31, CD33, CD34, CD45, CD117 and MHC class II molecules. The positive rate is below 5%. Consistent with the foregoing results. (Figure 6)

The cell cycle was measured by flow cytometry. The cells were fixed with 80% cold ethanol at 4 ° C for 1 h, washed twice with PBS, resuspended in 0.5 ml PBS, added with RnaseA (100 g/ml), and incubated at 37 °C for 30 minutes. Stained with propidium iodide (Sigma, 5 μg/ml) before the measurement. Cell cycle was analyzed with ModiFit software (Becton Dickinson). Cell cycle assays were performed at passages 3 and 7 respectively. The results showed that most of the spindle cells obtained in the three cultures were in the G0/G1 phase, accounting for more than 85%, while the proportion of cells in the G2-M phase and the S phase was very low. Subculture period remains stable Set. The results indicated that peripheral blood serum and cord blood serum culture medium did not change the basic biological indicators such as morphology, immunophenotype and cell cycle of pMSCs during the longer period of culture. The cell cycle difference of pMSCs in the three cell culture media was not significant and was not statistically significant. (Figure 7)

2. Study on the multi-directional differentiation ability of pMSCs

The pMSCs cultured in the third and seventh generations were cultured in three mediums, and induced to differentiate into osteoblasts, fat and endothelium by conventional methods. The differentiation ability of pMSCs in peripheral blood serum and cord blood serum was found. There was no significant difference in the differentiation ability of pMSCs with fetal bovine serum system. (Fig. 8-1, Fig. 8-2, Fig. 8-3)

3. Comparison of growth curves of pMSCs in three media

The 3rd and 7th generation pMSCs were cultured in three mediums, and the cells were seeded in a 24-well plate at ⁵ ×10 ³ cells/well, then 3 wells were digested every 24 hours, and the cells were collected and used. 0.4% of the trypan blue counted live cells and then plotted the growth curve. The results showed that pMSCs cultured in peripheral blood serum medium proliferated faster, the doubling time was about 28.5 hours; the doubling time of pMSCs cultured in fetal bovine serum medium was about 29.4 hours, and the doubling time of pMSCs in umbilical cord serum medium was about 30. Hours, the differences between the three were not statistically significant. (Fig. 9) Example 4: Application of human pMSCs to inhibit tumor cell proliferation

1. Human pMSCs expression Nanog gene detection

The human pMSCs prepared by the method described in the present application specifically expressed Nanog and Dkk-1 genes, and were detected by RT-PCR, and the results showed that the Nanog and Dkk-1 genes were positive (Fig. 10).

2. Inhibition of pMSCs on the proliferation of three tumor cell lines

With ³ H-thymidine ^{^{(3 H-thymidine, 3 H}} -TDR) incorporation assay tumor cell proliferation. The 30 Gy-irradiated pMSCs were co-cultured with cells of three human hematological tumor cell lines (K562, HL60, MCF7) at a ratio of 1:10. The results showed that: After co-culture with pMSCs, the proliferation ability of the three tumor cells was compared with that of the culture alone. The decrease was observed in which K562 decreased by 77%, HL60 decreased by 80%, and MCF7 decreased by 56%. The difference was statistically significant (t test, p<0.05) (Fig. 11-1).

3. Inhibition of pMSCs on the cell cycle of k562

The cell cycle of k562 was detected by flow cytometry. The pMSCs irradiated with 30 Gy were co-cultured with k562 at a ratio of 1:10. The results showed that the proportion of cells in the G0/G1 phase (62.07 ± 5.8% vs. 45.23 ± 6.9) was compared between the cocultured K562 (Fig. 11-2, group 2) and the culture alone (Fig. 11-2, group 1). %, p<0.05) increased significantly.

To investigate the mechanism of inhibition of pMSCs on the proliferation of tumor cell line k562, the transwell assay was used to detect the effect of soluble factors, and the neutralizing antibody assay was used to determine the soluble factor class. The results showed that after transwell, the proportion of cells in the G0/G1 phase (63.65 8.43%) was compared between the co-cultured K562 (Fig. 11-2, group 4) and the culture alone (Fig. 11-2, group 1). There was a significant increase in 45.23±6.9%, p<0.05). However, there was no statistical difference in k562 cell cycle between transwell and untranswelled co-culture (t test, p>0.05). The results showed that the inhibitory effect of pMSCs on k562 cells was through the secretion of soluble factors. When DMSC neutralizing antibody was co-cultured with pMSCs and K562, the cell cycle of K562 inhibition was restored to 49.22±1.21% (Fig. 11-2, group 3), demonstrating that Dkk acts as a soluble secretory factor. 4. Detection of inhibition of K562 cell cycle by pMSCs after interference of Nanog and DKK-1 genes, respectively

The method of transfection with siRNA-retroviral vector specifically targets RNA interference of Nanog and DKK-1 genes of pMSCs. The expression of the target gene in the cells after transfection of the interference vector was detected by Real-time RT-PCR and western blot. The pMSCs (SiN group) in which the Nanog gene was interfered and the pMSCs (SiD group) in which the DKK-1 gene was interfered were co-cultured with k562 at a ratio of 1:10 after 30 Gy irradiation; the control group was a negative control for transfection. The interfering empty vector pMSCs (SiM group) were co-cultured with k562 at a ratio of 1:10 after 30 Gy irradiation. The results showed that at the RNA level, the level of nanog mRNA in the pMSCs of the SiN group was 72% lower than that of the normal pMSCs (Fig. 11-3A). The level of DKK1 mRNA in the pMSCs of the SiD group was 78% lower than that of the normal pMSCs (Fig. 11-3B). ), the difference was statistically significant (t test, p<0.05); at protein level Above, the expression levels of Nanog and DKK-1 in the experimental group were significantly lower than those in the normal pMSCs and SiM groups (Fig. 11-3C). It can thus be determined that the interference vector exerts a significant and stable down-regulation effect. Among them, while the expression of Nanog gene was down-regulated in pMSCs that interfered with Nanog gene, the expression of DKK-1 gene was also down-regulated (Fig. 11-3B, C). The results indicated that Nanog expressed in pMSCs as a transcription factor may be involved in DKK- 1 The transcriptional regulation of genes, which in turn affects the expression of their proteins.

The cell cycle of k562 was detected by flow cytometry. The results showed that the proportion of k562 cells in the G0/G1 phase (52.09±3.36 vs. 62.02±4.36, p<0.05) was reduced in the SiD group compared with the SiM group. The SiN group was in the G0/G1 phase at the K562 compared with the SiM group. The proportion of cells (46.32±4.77 vs. 62.02±4.36, p<0.05) was significantly reduced (Fig. 11-4). After the Nanog and DKK-1 genes were disrupted, the effect of pMSCs on the tumor cell cycle was significantly attenuated.

5. Detection of the effect of pMSCs on the accumulation of β-catenin in Wnt signaling pathway in K562

The activation of Wnt signaling pathway allows β-catenin to accumulate in cells, and the accumulation of β-catenin into the nucleus promotes the rapid entry of cells from the G1 phase into the S phase, thereby promoting tumor cell proliferation. The accumulation of β-catenin protein in k562 cells was detected by westrn blot. The pMSCs interfered with by the Nanog gene and the pMSCs interfered with the DKK-1 gene were co-cultured with k562 at a ratio of 1:10 after 30 Gy irradiation; the control group was a pMSCs transfected with a negative control interference vector. After 30 Gy irradiation, co-culture with k562 and normal k562 cultured alone were performed at a ratio of 1:10.

The results showed that the accumulation of β-catenin in k562 and pMSCs was decreased after co-culture, but the accumulation of β-catenin in k562 cells was increased after co-culture of k562 and Nanog or Dkk gene-interfering pMSCs (Fig. 11- 5), indicating that pMSCs inhibit the wnt pathway in k562 cells by expressing Nanog, Dkk gene.

6. Detection of the effect of pMSCs on the expression of cell cycle-associated proteins in K562

There are many important regulatory proteins in the cell cycle from the G1 phase to the S phase. Real-time RT-PCR was used to detect the expression of cell cycle regulatory genes c-myc, CyclinD2 and cell cycle inhibitory genes P21 and P27 in k562 cells co-cultured with pMSCs. The pMSCs (SiN group) in which the Nanog gene was interfered and the pMSCs (SiD group) in which the DKK-1 gene was interfered were co-cultured with k562 at a ratio of 1:10 after 30 Gy irradiation; The pMSCs (SiM group) which were transfected with the negative control interference vector were irradiated with 30 Gy, co-cultured with k562 at a ratio of 1:10, and normal k562 cultured alone. The results showed that the expression of P21 and P27 was increased in the SiM group compared with the normal k562 group, and the expression of the regulatory genes c-myc and CyclinD2 was decreased. The expression of the inhibitory genes P21 and p27 was decreased in the SiN group and the SiD group compared with the SiM group. (Fig. 11-6A, B), the expression of the regulatory gene c-myc, CyclinD2 was increased (Fig. 11-6C, D), indicating that pMSCs inhibit the proliferation of k562 cells by expressing Nanog, Dkk gene.

Claims

Claims:

I. A medium for culturing primitive mesenchymal stem cell pMSCs derived from human adult tissues, comprising a cell basal medium and human serum, the final concentration of said human serum being 10-100 ml L.

2. The medium according to claim 1, wherein the final concentration of the human serum is

10-30ml/L.

3. The medium according to claim 1, wherein the human serum has a final concentration of 20 ml/L.

The medium according to claim 1 or 3, wherein the human serum is human peripheral or cord blood serum.

The medium according to any one of claims 1 to 3, further comprising human albumin, epidermal growth factor EGF and platelet-derived growth factor PDGF, wherein the final concentration of the human serum albumin is 5-20 mg /ml, the final concentration of the epidermal growth factor is 1-100 ng/ml, and the final concentration of the platelet-derived growth factor is 1-100 ng/ml.

6. The medium according to claim 5, wherein the final concentration of the human serum albumin is 5-10 mg/ml, the final concentration of the epidermal growth factor is 10-30 ng/ml, and the platelet-derived growth The final concentration of the factor is 10-30 ng/ml.

7. The medium according to claim 6, wherein the final concentration of the human serum albumin is 10 mg/ml, the final concentration of the epidermal growth factor is 10 ng/ml, and the final concentration of the platelet-derived growth factor. It is 10 ng/ml.

The medium according to claim 1 or 3, wherein the human adult tissue is

9. A method of in vitro culture of primitive mesenchymal stem cell pMSCs derived from human adult tissue, the method comprising the steps of:

1) suspending human adult tissue mononuclear cells in a medium according to any one of claims 1-3 as a primary cell suspension;

2) inoculating the primary cell suspension at a seeding density of ¹ ×10 ⁶ cells/ml in a plastic petri dish containing the medium according to any one of claims 1 to 3; 1-5%0 ₂ concentration, 5 % C0 ₂ concentration and 37 ° C, cultured in a saturated humidity incubator; repeated adherent culture 3-4 times to obtain primitive mesenchymal stem cells of human adult tissues.

10. The method of claim 9, wherein the human adult tissue is bone marrow.

I I. The method according to claim 9 or 10, wherein the 0 ₂ concentration is 2%

12. Raw mesenchymal stem cells obtained by the method of claim 9 or 10.

The primordial mesenchymal stem cell according to claim 12, which has an immunophenotype of Flkl-positive and expresses a Nanog gene.

The use of the primordial mesenchymal stem cells according to claim 12 for the preparation of a medicament for inhibiting proliferation of tumor cells.

A pharmaceutical composition comprising the primary mesenchymal stem cell according to claim 11.

16. An adult primitive mesenchymal stem cell culture PEPSC, deposited under the accession number CGMCC No. 2152, deposited on September 4, 2007 at the General Microbiology Center of the China Collection of Microorganisms, including primitive mesenchymal stem cells.

The adult primordial mesenchymal stem cell culture PEPSC according to claim 16, wherein the primordial mesenchymal stem cells have an immunophenotype of Flkl-positive and express a Nanog gene.

18. Use of an adult primordial mesenchymal stem cell culture PEPSC according to claim 16 or 17 for the preparation of a medicament for inhibiting tumor cell proliferation.

A pharmaceutical composition comprising the adult original mesenchymal stem cell culture PEPSC according to claim 16 or 17.