CN101709289A - Method for inducing transformation of totipotent stem cells into mesenchymal stem cells - Google Patents

Abstract

The invention provides a method for inducing totipotent stem cells to become mesenchymal stem cells. The invention uses a simple culture method, uses fibroblast culture medium and a monoclonal sorting method to purify mesenchymal stem cells, and makes mesenchymal stem cells When the content is more than 95%, more than 10 ⁹ mesenchymal stem cells can be obtained; the beneficial effects of the present invention are mainly reflected in: (1) the cells obtained in the present invention have a good ability to expand the environment, and can be passed to 15 generations while maintaining their characteristics, and It has the ability to form bone, cartilage and tendon; (2) the method for obtaining cells obtained by the present invention is simple and efficient, and does not require separation and purification methods such as flow cytometry and magnetic beads, which greatly reduces the cost of obtaining mesenchymal stem cells from totipotent stem cells; (3) The cells of the present invention are suitable for defect repair of tissues such as bone, tendon, cartilage, and skin, and as seed cells for tissue engineering.

Description

A method for inducing totipotent stem cells to become mesenchymal stem cells

(1) Technical field

The invention relates to a method for inducing totipotent stem cells to become mesenchymal stem cells.

(2) Background technology

The damage of various tissues of the human body is very common, especially connective tissue damages such as bone, cartilage defect and tendon, ligament are more and more (accounting for 50% of sports injury), and skin damage is countless especially. Statistics show that in 2001, there were 408,000 cases of bone grafting in Europe, while there were 605,000 cases in the United States alone. At present, the global population over 65 years old is growing at a rate of 2-3% every year. And due to the improvement of people's material living standards, the change of lifestyle and the improvement of medical level, these have led to more demand and wider application of frontal tendons, ligaments, cartilage, bone grafts, and bone repair operations.

In my country, according to incomplete statistics, there are 3 million patients with bone defects or bone injuries caused by various traffic accidents, orthopedic diseases and other factors, and tens of millions of people with unsound bones. The total bone market is at least 50 million yuan. Tendon injury is one of the most common sports injuries, accounting for more than 50% of it. After the tendon ruptures, the corresponding joint loses its function of movement. Statistics show that there are at least tens of millions of tendon injuries per 200 million people a year. In addition, due to changes in lifestyle, the cases of hand tendon injuries caused by long-term use of computer keyboards and sending mobile phone text messages are also increasing at a rate of 5% per year.

At present, the injuries of clinical sports organs and tissue defects such as skin are mainly repaired and strengthened by autologous/allogeneic tissues, or repaired by non-degradable biomaterials. But these treatments all have their inherent drawbacks. For example, the transplantation of autologous tissue needs to sacrifice the function of the donor area, and the supply is very limited; the source of allogeneic tissue is difficult and there may be immune and pathological problems.

At present, the method of tissue engineering is considered to be the most promising method for regenerating sports organ injuries and tissue defects such as skin. There have been many studies exploring the application of tissue engineering methods to regenerate defective tissues. According to the report of the authoritative journal Lancet, tissue engineering trachea has been successfully applied to patients with tracheal defect recently, which provides a basis for the application of tissue engineering in tissue regeneration. Tissue engineering is a method of compounding seed cells, materials and growth factors to construct functional tissues in vivo or in vitro for repairing or regenerating defective tissues. Seed cells are an important basic element in tissue engineering, and are the basis for the constructed tissue to have functions and to regenerate defective tissue in vivo.

The cells currently used for connective tissue regeneration mainly include adult mesenchymal stem cells derived from various tissues, skin fibroblasts and other differentiated mature somatic cells and totipotent stem cells (embryonic stem cells and induced pluripotent stem cells (iPS) derived from somatic cells) . However, these cell sources all have their inherent defects: 1) The differentiated mature somatic cells have only limited ability to differentiate and expand. 2) Mesenchymal stem cells have a strong ability to differentiate and expand, but they have not yet achieved the ability to fully regenerate embryonic tissue. 3) Embryonic stem cells are the earliest embryonic cells with pluripotent differentiation ability and are considered to have the most regenerative potential. However, due to their strong tumorigenicity, undifferentiated embryonic stem cells cannot be used for repair. 4) iPS is similar to embryonic stem cells, and undifferentiated iPS cannot be used for repair. But it can be derived from autologous cells, so it has wider application prospects. However, in view of the tissue regeneration potential of embryonic stem cells and iPS, if embryonic stem cells or iPS are induced to differentiate into connective tissue stem cells or precursor cells (mesenchymal stem cells and bone, cartilage, tendon and other progenitor cells) to remove their tumorigenicity, The totipotent stem cells can be applied to tissue engineering repair. At present, the method of inducing totipotent stem cells into mesenchymal stem cells is cumbersome and costly, and cells need to be purified by methods such as flow cytometry. Therefore, finding a simple and low-cost method to induce totipotent stem cells to become mesenchymal stem cells is a prerequisite for the application of totipotent stem cells in connective tissue engineering.

(3) Contents of the invention

The purpose of the present invention is to provide a simple and efficient method for inducing totipotent stem cells to become mesenchymal stem cells.

The technical scheme adopted in the present invention is:

A method for inducing totipotent stem cells to become mesenchymal stem cells, the method comprising:

(1) Culture the totipotent stem cells on the mouse MEF feeder layer treated with mitomycin (see "Cultivation of Mouse Embryonic Stem Cells - Preparation of Feeder Layer" for the treatment method, Chinese Journal of Cell Biology 2009, 31(2): 291-292), cultured with cell culture medium I for 3-5 days, and after the totipotent stem cells were congested, trypsin-EDTA (that is, trypsin/EDTA digestion solution, trypsin concentration 0.01-0.05%, EDTA Concentration 0.01～0.05%, w/v) is digested into single cell suspension; The final concentration composition of described cell culture medium I is as follows: 5～20% (w/v, mass volume percent concentration, certain component concentration 1% means 100mL The culture solution contains 1g of this component) KO serum substitute, 1-5mM L-glutamine, 0.5-5.0% (w/v) non-essential amino acids, 5-20ng/ml basic fibroblast growth factor 2, 50 ～200U/ml penicillin, 50～200U/ml streptomycin, the solvent is KO-DMEM;

(2) In step (1), the single cell suspension is planted on a petri dish at a density of 100-1000cell/cm ² , and subcultured in cell culture medium II; the composition of the final concentration of the cell culture medium II is as follows: 10-30% (w/v) Fetal bovine serum, 50-200U/ml penicillin, 50-200U/ml streptomycin, the solvent is low-sugar DMEM;

(3) The third-generation cells obtained from the culture in step (2) are planted on a petri dish at a density of 2-10 cells/cm ² , and cultured in cell culture medium III until monoclonal cells grow out to obtain the mesenchyme Stem cells; the composition of the final concentration of the cell culture medium III is as follows: 5-20% (w/v) fetal bovine serum, 50-200 U/ml penicillin, 50-200 U/ml streptomycin, and the solvent is low-sugar DMEM.

The cells are fibroblast-like cells induced and differentiated from pluripotent stem cells and planted at a low density in a specific medium. They have the ability to differentiate into bone, cartilage, tendon and fat. Compared with adult cells, they also have good Amplification ability, can be subcultured to more than 15 generations while maintaining the original characteristics.

The embryonic stem cells can be commercially available, such as H1 and H9 cell lines from WiCell Inc, or obtained from discarded embryos. The specific method of obtaining embryonic stem cells from embryos is as follows: 1. Collect low-quality embryos on the third day of the IVF/ICSI treatment cycle, and when they are cultured in vitro for 5 to 6 days, the inner cell mass can be separated and cultured on mouse embryonic fibroblasts After 9 to 15 days, the grown inner cell mass was blown away or digested with trypsin, and then cultured on mouse fibroblasts. 2. Pick out single-cell clone groups with uniform undifferentiated morphology, blow them into small groups of 50-100 cells, and then culture them. 3. Repeat this until a line is formed.

Identification of mesenchymal stem cells: 1) Observation of cell morphology. Cell adherence is the first day of differentiation, and the morphology of adherent cells is observed every day. After screening monoclonal cells, the proportion of fibroblast-like cells is >95%. 2) Cell phenotype identification, stromal cell phenotype positive (CD44, CD90, CD105, CD106), hematopoietic phenotype negative (CD34), mesenchymal cells accounted for more than 95%. 3) Ability to proliferate, differentiate and tissue differentiate The cells have the ability to differentiate into bone, cartilage and fat.

Preferably, the final concentration of cell culture medium I in the step (1) is composed as follows: 10% KO serum substitute, 2mM L-glutamine, 1% non-essential amino acids, 10ng/ml basic fibroblast growth factor 2, 100U/ml penicillin, 100U/ml streptomycin, the solvent is KO-DMEM.

Preferably, the composition of the final concentration of the cell culture medium II in the step (2) is as follows: 20% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, and the solvent is low-sugar DMEM.

Preferably, the composition of the final concentration of the cell culture medium III in the step (3) is as follows: 10% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, and the solvent is low-sugar DMEM.

The present invention is a further improvement on the basis of previous studies, using a simple culture method, using a fibroblast culture medium and a monoclonal sorting method to purify mesenchymal stem cells, so that the content of mesenchymal stem cells is above 95%. Obtain more than 10 ⁹ mesenchymal stem cells, so as to obtain enough connective tissue seed cells. This technology will promote the application of totipotent stem cells in tendon, bone, cartilage and skin tissue engineering.

The beneficial effects of the present invention are mainly reflected in:

(1) The cells obtained in the present invention have good environment-expanding ability, can be passed to 15 generations and maintain their characteristics, and have the ability to form bone, cartilage and tendon;

(2) The method for obtaining cells obtained in the present invention is simple and efficient, and does not require separation and purification methods such as flow cytometry and magnetic beads, which greatly reduces the cost of obtaining mesenchymal stem cells from totipotent stem cells;

(3) The cells of the present invention are suitable for defect repair of tissues such as bone, tendon, cartilage, and skin, and as seed cells for tissue engineering.

(4) Description of drawings

Fig. 1 is primary cell of totipotent stem cell adherent differentiation;

Figure 2 shows the morphology of seed cells derived from totipotent stem cells, and the cell morphology after induction has a high degree of uniformity;

Figure 3 shows that the seed cells derived from totipotent stem cells have a mesenchymal stem cell phenotype;

Figure 4 shows that seed cells derived from totipotent stem cells have three-line differentiation ability similar to mesenchymal stem cells: A: bone differentiation ability, B: cartilage differentiation ability, C: fat differentiation ability, D: tendon differentiation ability;

Figure 5 is the repair of tendon with seed cells derived from totipotent stem cells; A: Seed cells were planted in fibrin glue; B: Seed cells derived from totipotent stem cells repaired patellar tendon defect;

Figure 6 shows the tendon repaired by seed cells derived from totipotent stem cells; A: tendon-like structure appears at the defect site; B: continuous mature collagen fibers appear at the defect site; T-normal tendon, J-repair junction, N-repair site;

Figure 7 is the repair of cartilage by seed cells derived from totipotent stem cells; A: cartilage defect repaired by seed cells derived from totipotent stem cells; B: histology of cartilage defects repaired by seed cells derived from totipotent stem cells.

Figure 8 shows the repair of large bone defects with seed cells derived from totipotent stem cells; A: seed cells derived from totipotent stem cells were planted in demineralized bone to construct tissue engineered bone; B: tissue engineered bone repaired large bone defects.

Figure 9 shows that seed cells derived from totipotent stem cells are used as feeder cells of human embryonic stem cells: human embryonic stem cells can grow normally on seed cells derived from totipotent stem cells.

(5) Specific implementation methods

The present invention is further described below in conjunction with specific embodiment, but protection scope of the present invention is not limited thereto:

Example 1 (the reagents in this example were purchased from Invitrogen Company except for special instructions):

1) The culture wells are coated with 0.1% (w/w) gelatin, and the totipotent stem cells (human embryonic stem cells, obtained from discarded embryos, see the content of the invention for the method. The discarded embryos are donated after the consent of the embryo provider himself and his family) Inoculate on mouse MEF feeder layer treated with mitomycin, culture with cell culture medium I at 37°C, 5% CO ₂ (the remaining 95% is air) for 3-5 days, and use when the cells are congested Digest with 0.025% trypsin-0.05% EDTA (Invitrogen) and blow off to obtain a single cell suspension.

MEF feeder layer treatment:

1. Take a new culture bottle, add 0.1% Gelatin (SigmaG2500) solution to cover the pretreatment for 30 minutes, and suck off the Gelatin solution before use.

2. Take the MEF cells that need to be treated, add Mitomycin C (Sigma M0503) to the MEF culture medium at a working concentration of 10mg/ml, and mix well.

3. Put it in the incubator for 3 hours.

4. Absorb the waste liquid.

5. Wash 5 times with PBS.

6. Add 0.025% Trypsin-EDTA for digestion. Observe under a microscope, when a small amount of cells float and there are cracks in the adherent cell layer, blow and wash the bottom of the bottle 5-6 times with a pipette.

7. Immediately add an appropriate amount of MEF complete culture medium to stop digestion.

8. Transfer the cell suspension to a centrifuge tube and centrifuge at 1000r/min for 5min. Aspirate and discard supernatant.

9. The treated MEF cells were resuspended in an appropriate amount of culture medium and counted, and evenly spread on a gelatin-treated culture flask at a density of 3.0×10 ⁴ cells/cm ² (MEF).

10. Put it in the incubator overnight to make it adhere to the wall.

The final concentration of cell culture medium I was composed as follows: 10% (w/v) KO serum replacement (Invitrogen), 2 mM L-glutamine, 1% (w/v) non-essential amino acids (Invitrogen), 10 ng/ml alkaline Fibroblast growth factor 2 (FGF2), 100 U/ml penicillin, 100 U/ml streptomycin, the solvent is KO-DMEM.

MEF complete culture medium: 10% (w/v) fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, solvent is high sugar DMEM.

2) The single cell suspension is planted on a culture dish at a density of 500-600cell/cm ² , and subcultured in cell culture medium II; the final concentration of the cell culture medium II is composed as follows: 20% (w/v) fetal bovine Serum, 100U/ml penicillin, 100U/ml streptomycin, the solvent is low-sugar DMEM;

3) The third-generation cells (cells with relatively uniform fibroblast-like cells) obtained from step 2) were planted on a petri dish at a density of 6-10 cells/cm ² , and cultured in cell culture medium III until the monoclonal cells grew. The mesenchymal stem cells were obtained; the final concentration of the cell culture medium III was composed as follows: 20% (w/v) fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, and the solvent was low-sugar DMEM. The cells were seen as uniform fibroblast-like cells with a mesenchymal phenotype (Figure 1-3).

Example 2:

After the cells obtained in Example 1 were cultured to confluence, 2.5×10 ⁷ cells (50ul, 5×10 ⁸ /ml) were planted on chitosan gel to construct tissue-engineered cartilage (see FIG. 4B ).

Example 3:

After the cells obtained in Example 1 were cultured to full length, 50ug/ml vitamin C was added to culture for 14 days to form a cell sheet, and the cell sheet was cultured for 2 weeks under static tension stimulation (stretching 10%), observed by histology and electron microscope, It can be seen that the cells are arranged regularly in the mechanical direction, constituting the tissue engineered tendon in vitro (see Figure 4D).

Example 4:

The cells obtained in Example 1 were implanted into rats to repair the patellar ligament defect, and 5×10 ⁵ cells (20ul, 2.5×10 ⁷ /ml) were planted in Fibrin gel, and planted in the patellar ligament of rats. After 4 weeks, it was found that the tissue had grown into the defect site, forming functional tendon tissue (see Figures 5 and 6).

Example 5:

The cells obtained in Example 1 were implanted into rats to repair knee articular cartilage defects. 5×10 ⁵ cells (20ul, 2.5×10 ⁷ /ml) were placed in collagen gel. After 4 weeks, the tissue grew well and could form functions Sexual cartilage tissue, no obvious inflammatory reaction (see Figure 7).

Embodiment 6:

The cell sheet obtained in Example 3 was combined with demineralized bone to construct tissue engineered bone (see FIG. 8A ), which is used to repair radial defect (see FIG. 8B ), which can promote the growth of new bone tissue.

Embodiment 7:

The cells obtained in Example 1 were treated with mitomycin c as human embryonic stem cell feeder cells (see FIG. 9 ): human embryonic stem cells can grow normally.

Claims (4)

1. method of inducing myeloid-lymphoid stem cell to become mescenchymal stem cell, described method comprises:

(1) myeloid-lymphoid stem cell is incubated on the mouse MEF feeder layer that mitomycin handled, cultivated 3～5 days with cell culture medium I, myeloid-lymphoid stem cell covers with the back and is digested to single cell suspension with pancreas enzyme-EDTA; Described cell culture medium I final concentration is composed as follows: 5～20%KO serum substitute, 1～5mM L-glutaminate, 0.5～5.0% non-essential amino acid, 5～20ng/ml Basic Fibroblast Growth Factor 2,50～200U/m1 penicillin, 50～200U/ml Streptomycin sulphate, solvent are KO-DMEM;

(2) step (1) single cell suspension is with 100～1000cell/cm ²Density is planted on culture dish, the cultivation of going down to posterity in cell culture medium II; Described cell culture medium II final concentration is composed as follows: 10～30% foetal calf serums, and 50～200U/ml penicillin, 50～200U/ml Streptomycin sulphate, solvent are low sugar DMEM;

(3) step (2) is cultivated the third generation cell of acquisition again with 2～10cell/cm ²Density is planted on culture dish, cultivates to grow until monoclonal cell in cell culture medium III, promptly gets described mescenchymal stem cell; Described cell culture medium III final concentration is composed as follows: 5～20% foetal calf serums, and 50～200U/ml penicillin, 50～200U/ml Streptomycin sulphate, solvent are low sugar DMEM.

2. the method for claim 1, it is characterized in that cell culture medium I final concentration is composed as follows in the described step (1): the 10%KO serum substitute, the 2mM L-glutaminate, 1% non-essential amino acid, 10ng/ml Basic Fibroblast Growth Factor 2,100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are KO-DMEM.

3. the method for claim 1 is characterized in that cell culture medium II final concentration is composed as follows in the described step (2): 20% foetal calf serum, and 100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are low sugar DMEM.

4. the method for claim 1 is characterized in that cell culture medium III final concentration is composed as follows in the described step (3): 10% foetal calf serum, and 100U/ml penicillin, 100U/ml Streptomycin sulphate, solvent are low sugar DMEM.

Images