CN107083367B - Culture medium, application thereof and method for preparing mesenchymal stem cells from urine cells - Google Patents

Culture medium, application thereof and method for preparing mesenchymal stem cells from urine cells Download PDF

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CN107083367B
CN107083367B CN201710533052.9A CN201710533052A CN107083367B CN 107083367 B CN107083367 B CN 107083367B CN 201710533052 A CN201710533052 A CN 201710533052A CN 107083367 B CN107083367 B CN 107083367B
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cells
culture medium
mesenchymal stem
stem cells
urine
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CN107083367A (en
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黄燕飞
车七石
赵澎
刘少辉
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Guangzhou Rainhome Pharm and Tech Co Ltd
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Abstract

The invention relates to the technical field of stem cells and genetic engineering, in particular to a culture medium and application thereof, and a preparation method of mesenchymal stem cells. The method for inducing the urine cells to become the mesenchymal stem cells by the culture medium provided by the invention has the advantages that the urine cells are wide in source and are not limited by ethics, and the urine cells sequentially form IPS cells, embryoid bodies and mesenchymal stem cells by the method provided by the invention. All induction can be completed within 20-25 days, and the obtained cells meet the characteristics of mesenchymal stem cells through detection, and are large in number and high in survival rate.

Description

Culture medium, application thereof and method for preparing mesenchymal stem cells from urine cells
Technical Field
The invention relates to the technical field of stem cells and genetic engineering, in particular to a culture medium and application thereof, and a preparation method of mesenchymal stem cells.
Background
With the rise of tissue engineering and clinical transplantation medicine, research on seed cells is receiving more and more attention. Mesenchymal Stem Cells (MSCs) are a class of pluripotent stem cells that are present in a variety of tissues, such as bone marrow, umbilical cord blood and tissue, placental tissue, adipose tissue, and the like. It has the ability to proliferate highly, renew itself and has the potential to differentiate divergently. Can differentiate into cells of cartilage, bone, skeletal muscle, tendon, fat, dermis, nerve and renal parenchyma under different induction conditions. Therefore, mesenchymal stem cells are often used as seed cells.
Clinical research on mesenchymal stem cells has been carried out in many countries, more than 60 clinical trials have been approved in the united states, and with the increasing maturity of mesenchymal stem cells and related technologies thereof, many clinical trials have been approved in China, and the research and development stage of the mesenchymal stem cell core technology has entered. At present, clinical trials have demonstrated that MSCs inhibition can be used for tissue repair and for the treatment of mesenchymal genetic defects, such as: hematological diseases, cardiovascular diseases, liver cirrhosis, neurological diseases, repair of meniscal resection injuries of the knee joint, autoimmune diseases, and the like. The characteristics of the mesenchymal stem cells determine that the mesenchymal stem cells have good clinical application prospect.
At present, the main sources of mesenchymal stem cells are bone marrow and umbilical cord tissues, the number of the mesenchymal stem cells is obviously reduced along with the aging of the mesenchymal stem cells, the proliferation and differentiation capacity is greatly declined, the quality control is not easy in the preparation process, and the transplantation of allogenic cells can cause immune reaction and has great harm to human bodies. Although the immunity of umbilical cord mesenchymal stem cells is low, the storage of umbilical cord blood is expensive. The number of stem cells derived from other tissues is very small, and isolation and culture are also very difficult. Therefore, further research on preparation methods of mesenchymal stem cells remains a hot spot of research in this field at present.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a culture medium, use thereof, and a method for preparing mesenchymal stem cells. The culture medium provided by the invention can reprogram urine cells into IPS cells, induce the IPS cells into embryoid bodies and then directionally differentiate the embryoid bodies into mesenchymal stem cells.
The invention provides a pseudo-embryo body induction culture medium, which comprises a basic culture medium and:
Figure BDA0001339850280000021
in some embodiments, the embryoid body-inducing medium consists of a basal medium and the following components:
Figure BDA0001339850280000022
in the invention, FBS, L-glutamine, β -mercaptoethanol, BMP-4, VEGF and insulin are added into a basic culture medium, wherein the basic culture medium can induce the IPS cell to be transformed into an embryoid body, and the DMEM culture medium is used as the basic culture medium.
The embryoid body culture medium provided by the invention comprises a basic culture medium and:
Figure BDA0001339850280000023
in some embodiments, the embryoid body medium consists of a basal medium and the following components:
Figure BDA0001339850280000024
Figure BDA0001339850280000031
the ITS is insulin transferrin sodium selenite, the EGF is epidermal growth factor, the invention adds FBS, ITS, sodium pyruvate, EGF, L-glutamine and β -mercaptoethanol in a basic culture medium, and the obtained culture medium can be used for culturing embryoid bodies.
The mesenchymal stem cell induction culture medium provided by the invention comprises a basic culture medium and:
Figure BDA0001339850280000032
in some embodiments, the mesenchymal stem cell induction consists of a basal medium and the following components:
Figure BDA0001339850280000033
the bFGF is a basic fibroblast growth factor, the FBS, the insulin, L-glutamine, the bFGF and dexamethasone are added into a basic culture medium, and the obtained culture medium can induce and differentiate the embryoid bodies into mesenchymal stem cells.
The mesenchymal stem cell culture medium provided by the invention comprises a basic culture medium and:
Figure BDA0001339850280000034
in some embodiments, the mesenchymal stem cells consist of a basal medium and the following components:
Figure BDA0001339850280000035
the method comprises the steps of adding FBS, EGF, HGF, bFGF, PDGF and TGF- β into a basal medium, wherein the obtained culture medium can be used for culturing mesenchymal stem cells.
The invention also provides a method for inducing the IPS cell to form an embryoid body, which is to induce the IPS cell by using an embryoid body induction culture medium; the induction time is 3-10 days.
In some embodiments, the induction time is 7-10 days. The induction conditions were 37 ℃, saturated humidity, 5% CO2Density of induction inoculation of 1 × 104～0.5×104cell/m L the induction was performed in a plate coated with agarose at a mass fraction of 0.1% to 0.02%, during the culture, the medium was changed daily.
Experiments show that when the method is used for inducing the IPS cells, embryoid bodies begin to appear 3-4 days later, and the growth amount of the embryoid bodies cultured for 7-10 days reaches the highest peak.
The invention also provides a method for inducing the embryoid bodies to differentiate into the mesenchymal stem cells, which comprises the following steps: inoculating the embryoid-like body into an embryoid-like body culture medium, culturing for 10-14h, and inducing for 5-7 days by using a mesenchymal stem cell induction culture medium to obtain the mesenchymal stem cell.
In the present invention, the culture in the embryoid body-like medium is carried out on a gelatin-coated plate. The culture conditions were 37 ℃ and saturated humidity, 5% CO2. And after 10-14h, the embryoid body adheres to the wall, and the mesenchymal stem cells are replaced for induction culture. And (5) inducing the formation of mesenchymal stem cells after 5 to 7 days.
The expression condition of the gene in the mesenchymal stem cell is detected by RT-PCR, the expression quantity of NAONG and OCT-4 in the mesenchymal stem cell is hardly expressed, which indicates that the induced mesenchymal stem cell loses the expression of the pluripotent stem gene, and the mesenchymal stem cell belongs to mesoderm MSX-1, which is that the expression quantity of the mesoderm gene in the mesenchymal stem cell is obviously increased, and accords with the characteristics of the mesenchymal stem cell. Flow cytometry analysis shows that the expression of CD29, CD44, CD73, CD90 and CD105 in the mesenchymal stem cells is positive, and the expression of CD34 and CD45 is negative, which accords with the expression result of the mesenchymal stem cell surface marker.
The invention also provides a method for preparing mesenchymal stem cells from urine cells, which comprises the following steps:
introducing transcription regulatory factors OCT4, SOX2, NANOG, K L F4 and L IN28 into urine cells, and culturing to obtain IPS cells;
inducing IPS cells by using an embryoid induction culture medium, collecting the embryoid after 3-10 days, culturing for 10-14h by using the embryoid induction culture medium, and inducing for 5-7 days by using a mesenchymal stem cell induction culture medium to obtain the mesenchymal stem cells.
In the invention, the method also comprises the steps of proliferation and passage after the mesenchymal stem cells are obtained, and the mesenchymal stem cell culture medium is adopted for proliferation and passage.
In the invention, the preparation method of the urine cells comprises the following steps: urine was centrifuged, and the pellet was sterilized and cultured in REGM medium containing Primocin to obtain urine cells.
In the invention, the specific preparation method of the urine cells comprises the following steps:
step 1, collecting urine in a container containing penicillin/streptomycin double antibody, centrifuging for 10min at 400g, washing precipitate with PBS containing penicillin/streptomycin (5 m L penicillin/streptomycin is added to every 95m L PBS) once, and transferring to a culture plate coated with 0.1% gelatin;
step 2 culturing the ReGM medium containing Primocin (3. mu. L Primocin per 2m L REGM medium) at 37 ℃ with saturated humidity and 5% CO2(ii) a After the cells adhere to the wall, the culture medium is sucked off, washed once by PBS, and then subjected to liquid changing treatment; when the urine cell fusion degree reaches 80%, subculture is carried out.
After the transcriptional control factors OCT4, SOX2, NANOG, K L F4 and L IN28 are introduced into urine cells, the cells are cultured on a culture plate coated with gelatin by using a mixed culture medium (volume ratio is 1:1) of REGM and MEF, the culture medium is replaced by an IPS culture medium mTesR on day 2 after the transcriptional control factors are introduced, the clone is continuously proliferated, the monoclonal antibody similar to the human embryonic stem cells IN shape is picked up and inoculated on the culture plate coated with gelatin after the transcriptional control factors are introduced on day 7, and the induced pluripotent stem cells (IPS cells) are obtained by culturing the culture medium mTesR.
IPS cells are induced to form embryoid bodies after being digested into single cells by 0.03-0.3% collagenase, and then are induced to form mesenchymal stem cells.
The induction time of the IPS induction into the embryoid body is 7-10 days. The induction conditions were 37 ℃, saturated humidity, 5% CO2Density of induction inoculation of 1 × 104～0.5×104cell/m L the induction was performed in a plate coated with agarose at a mass fraction of 0.1% to 0.02%, during the culture, the medium was changed daily.
Culturing in embryoid body-like medium was performed on gelatin-coated plates. The culture conditions were 37 ℃ and saturated humidity, 5% CO2. And after 10-14h, the embryoid body adheres to the wall, and the mesenchymal stem cells are replaced for induction culture. And (5) inducing the formation of mesenchymal stem cells after 5 to 7 days.
The invention provides a culture medium and a method for inducing urine cells into mesenchymal stem cells by using the culture medium, wherein the urine cells have wide sources and are not limited by ethics. All induction can be completed within 20-25 days, and the obtained cells meet the characteristics of mesenchymal stem cells through detection, and are large in number and high in survival rate.
Drawings
Fig. 1 illustrates gene expression of mesenchymal stem cells prepared in example 1;
FIG. 2 shows the surface labeled antigen detection of the mesenchymal stem cells prepared in example 1.
Detailed Description
The invention provides a culture medium, application thereof and a preparation method of mesenchymal stem cells, and a person skilled in the art can realize the culture medium by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1
1. Preparation of the culture Medium
IPS induction medium (preparation medium a): REGM was mixed with MEF medium at a volume ratio (1: 1).
Embryoid body induction medium (configured medium B) 450m L DMEM, 50m L FBS, 5mM L-glutamine, 0.1mM β -mercaptoethanol, 50ng/m L BMP-4, 25ng/m L VEGF, 60pM insulin.
Embryoid body culture medium (configured culture medium C) 400m L DMEM, 100m L FBS, 5ug/ml insulin transferrin sodium selenite (ITS), 0.23mM sodium pyruvate, 1ng/ml Epidermal Growth Factor (EGF), 2mM L-glutamine, 0.1mM β -mercaptoethanol.
Mesenchymal stem cell induction medium (preparation medium D) is 450m L DMEM basal medium, 50m L FBS, 60pM insulin, 2mM L-glutamine, 75 ug/L bFGF, 3 × 10-8 mol/L dexamethasone.
Mesenchymal stem cell culture medium (configured culture medium E) 450m L DMEM basal medium, 50m L FBS, 10ng/m L hEGF, 10ng/m L bFGF, 3ng/m L HGF, 5ng/m L PDGF and 5ng/m L TGF- β.
2. Urine cell acquisition
1) 2m L penicillin/streptomycin double antibody was added to each collection cup.
2) Collecting urine, and storing the urine in a 4 deg.C refrigerator if the subsequent operation is not performed immediately, and completing the subsequent operation on the same day.
3) Six well plates were prepared for 1 well per urine, the wells were coated with 0.1% gelatin for more than 20min, and the liquid in the wells was aspirated before use.
4) The urine was poured into an appropriate number of 50m L centrifuge tubes and centrifuged at 400g for 10 min.
5) The supernatant was aspirated, leaving about 1-5m L per tube, and mixed into one centrifuge tube.
6) Adding PBS containing penicillin/streptomycin (PBS 95m L mixed with 5m L penicillin/streptomycin) about 10-30m L, and mixing gently.
7) Centrifuge 400g for 10 min.
8) The supernatant was aspirated until 0.5-1m L liquid remained.
9) The remaining liquid was added to the coated wells and 2m L REGM medium was added to 3 μ L Primocin.
10) The culture dish is placed in an incubator at 37 ℃ for culture, after the urine cells adhere to the wall (7 days), the culture medium is sucked off, washed once by PBS, and then subjected to liquid changing treatment.
11) When the urine cell fusion degree reaches 80%, subculture can be carried out.
3. Reprogramming urine cells to ips cells
1) Co-introducing various combinations of expression transcription regulatory factors OCT4, SOX2, NANOG, K L F4 and L IN28 or other transcription factors into urine cells, dividing the cells into 6-well plates coated with matrigel IN advance, and culturing with culture solution A;
2) on day 2 after transfection, the medium was changed to IPS medium mTesR, with a fresh medium change every day; continued propagation of clones
3) On day 7 after transfection, monoclonal antibodies similar in morphology to human embryonic stem cells were picked under microscopic observation and seeded in 12-well plates coated with matrigel, and cultured in medium mTesR to obtain induced pluripotent stem cells.
4. Induction of Ips cells into embryoid bodies
1) Digesting the prepared ips induced pluripotent stem cells into single cells by using 0.03% -0.3% collagenase IV, collecting the cells, washing the cells for three times by using PBS (phosphate buffer solution), and suspending the cells by using a culture solution B to prepare a suspension of 1 × 104-0.5 × 104 for later use.
2) The cell culture plate is coated with 0.1-0.02% agarose for 0.5-1 h.
3) Adding the cell suspension prepared in the step 1) into a culture plate coated with agarose, and performing liquid change treatment every day.
4) And when the embryoid appears after 3-4 days of culture, culturing for 7-10 days until the growth amount of the embryoid reaches the highest peak, collecting the embryoid suspension, standing for precipitation, and suspending by using the culture solution C for later use.
5. Directional differentiation of embryoid bodies into mesenchymal stem cells
1) The prepared embryoid body suspension was inoculated into a matrigel-coated plate.
2) Culturing the embryoid body for 10-14h, and adding culture solution D for induction culture when the embryoid body adheres to the wall.
3) The stem cells are formed after culturing for 5-7 days, and the culture solution E is replaced to continue culturing so as to continue proliferation.
Example 2
1. Preparation of the culture Medium
IPS induction medium (preparation medium a): REGM was mixed with MEF medium at a volume ratio (1: 1).
Embryoid body induction medium (preparation medium B) 450m L MEM, 50m L FBS, 1 mM-glutamine, 0.05mM β -mercaptoethanol, 10ng/m L BMP-4, 1ng/m L VEGF, and 1pM insulin.
Embryoid body culture medium (configured culture medium C) 400m L DMEM, 100m L FBS, 1ug/ml insulin transferrin sodium selenite (ITS), 0.1mM sodium pyruvate, 0.2ng/ml Epidermal Growth Factor (EGF), 1mM L-glutamine, 0.01mM β -mercaptoethanol.
Mesenchymal stem cell induction medium (preparation medium D) is 450m L DMEM basal medium, 50m L FBS, 1pM insulin, 1mM L-glutamine, 50 ug/L bFGF, 2 × 10-8 mol/L dexamethasone.
Mesenchymal stem cell culture medium (configured culture medium E) 450m L DMEM basal medium, 50m L FBS, 1ng/m L hEGF, 1ng/m L HGF, 1ng/m L bFGF, 2ng/m L PDGF and 1ng/m L TGF- β.
2. Urine cell acquisition
1) 2m L penicillin/streptomycin double antibody was added to each collection cup.
2) Collecting urine, and storing the urine in a 4 deg.C refrigerator if the subsequent operation is not performed immediately, and completing the subsequent operation on the same day.
3) Six well plates were prepared for 1 well per urine, the wells were coated with 0.1% gelatin for more than 20min, and the liquid in the wells was aspirated before use.
4) The urine was poured into an appropriate number of 50m L centrifuge tubes and centrifuged at 400g for 10 min.
5) The supernatant was aspirated, leaving about 1-5m L per tube, and mixed into one centrifuge tube.
6) Adding PBS containing penicillin/streptomycin (PBS 95m L mixed with 5m L penicillin/streptomycin) about 10-30m L, and mixing gently.
7) Centrifuge 400g for 10 min.
8) The supernatant was aspirated until 0.5-1m L liquid remained.
9) The remaining liquid was added to the coated wells and 2m L REGM medium was added to 3 μ L Primocin.
10) The culture dish is placed in an incubator at 37 ℃ for culture, after the urine cells adhere to the wall (7 days), the culture medium is sucked off, washed once by PBS, and then subjected to liquid changing treatment.
11) When the urine cell fusion degree reaches 80%, subculture can be carried out.
3. Reprogramming urine cells to ips cells
1) Co-introducing various combinations of expression transcription regulatory factors OCT4, SOX2, NANOG, K L F4 and L IN28 or other transcription factors into urine cells, dividing the cells into 6-well plates coated with matrigel IN advance, and culturing with culture solution A;
2) on day 2 after transfection, the medium was changed to IPS medium mTesR, with a fresh medium change every day; continued propagation of clones
3) On day 7 after transfection, monoclonal antibodies similar in morphology to human embryonic stem cells were picked under microscopic observation and seeded in 12-well plates coated with matrigel, and cultured in medium mTesR to obtain induced pluripotent stem cells.
4. Induction of Ips cells into embryoid bodies
1) Digesting the prepared ips induced pluripotent stem cells into single cells by using 0.03% -0.3% collagenase IV, collecting the cells, washing the cells for three times by using PBS (phosphate buffer solution), and suspending the cells by using a culture solution B to prepare 1 × 104-0.5×104The suspension of (a) is ready for use.
2) The cell culture plate is coated with 0.1-0.02% agarose for 0.5-1 h.
3) Adding the cell suspension prepared in the step 1) into a culture plate coated with agarose, and performing liquid change treatment every day.
4) And when the embryoid appears after 3-4 days of culture, culturing for 7-10 days until the growth amount of the embryoid reaches the highest peak, collecting the embryoid suspension, standing for precipitation, and suspending by using the culture solution C for later use.
5. Directional differentiation of embryoid bodies into mesenchymal stem cells
1) The prepared embryoid body suspension was inoculated into a matrigel-coated plate.
2) Culturing the embryoid body for 10-14h, and adding culture solution D for induction culture when the embryoid body adheres to the wall.
3) The stem cells are formed after culturing for 5-7 days, and the culture solution E is replaced to continue culturing so as to continue proliferation.
Example 3
1. Preparation of the culture Medium
IPS induction medium (preparation medium a): REGM was mixed with MEF medium at a volume ratio (1: 1).
Embryoid body induction medium (preparation medium B) 450m L MEM, 50m L FBS, 10 mM-glutamine, 0.5mM β -mercaptoethanol, 85ng/m L BMP-4, 50ng/m L VEGF, 100pM insulin.
Embryoid body culture medium (configured culture medium C) 400m L DMEM, 100m L FBS, 10ug/ml insulin transferrin sodium selenite (ITS), 0.65mM sodium pyruvate, 5ng/ml Epidermal Growth Factor (EGF), 5mM L-glutamine, 100mM β -mercaptoethanol.
Mesenchymal stem cell induction medium (preparation medium D) 450m L DMEM basal medium, 50m L FBS, 100pM insulin, 10mM L-glutamine, 150 ug/L bFGF, 5 × 10-8mol/L dexamethasone.
Mesenchymal stem cell culture medium (preparation medium E) 450m L DMEM basal medium, 50m L FBS, 100ng/m L hEGF, 100ng/m L bFGF, 50ng/m L HGF, 20ng/m L PDGF and 25ng/m L TGF- β.
2. Urine cell acquisition
1) 2m L penicillin/streptomycin double antibody was added to each collection cup.
2) Collecting urine, and storing the urine in a 4 deg.C refrigerator if the subsequent operation is not performed immediately, and completing the subsequent operation on the same day.
3) Six well plates were prepared for 1 well per urine, the wells were coated with 0.1% gelatin for more than 20min, and the liquid in the wells was aspirated before use.
4) The urine was poured into an appropriate number of 50m L centrifuge tubes and centrifuged at 400g for 10 min.
5) The supernatant was aspirated, leaving about 1-5m L per tube, and mixed into one centrifuge tube.
6) Adding PBS containing penicillin/streptomycin (PBS 95m L mixed with 5m L penicillin/streptomycin) about 10-30m L, and mixing gently.
7) Centrifuge 400g for 10 min.
8) The supernatant was aspirated until 0.5-1m L liquid remained.
9) The remaining liquid was added to the coated wells and 2m L REGM medium was added to 3 μ L Primocin.
10) The culture dish is placed in an incubator at 37 ℃ for culture, after the urine cells adhere to the wall (7 days), the culture medium is sucked off, washed once by PBS, and then subjected to liquid changing treatment.
11) When the urine cell fusion degree reaches 80%, subculture can be carried out.
3. Reprogramming urine cells to ips cells
1) Co-introducing various combinations of expression transcription regulatory factors OCT4, SOX2, NANOG, K L F4 and L IN28 or other transcription factors into urine cells, dividing the cells into 6-well plates coated with matrigel IN advance, and culturing with culture solution A;
2) on day 2 after transfection, the medium was changed to IPS medium mTesR, with a fresh medium change every day; continued propagation of clones
3) On day 7 after transfection, monoclonal antibodies similar in morphology to human embryonic stem cells were picked under microscopic observation and seeded in 12-well plates coated with matrigel, and cultured in medium mTesR to obtain induced pluripotent stem cells.
4. Induction of Ips cells into embryoid bodies
1) Digesting the prepared ips induced pluripotent stem cells into single cells by using 0.03% -0.3% collagenase IV, collecting the cells, washing the cells for three times by using PBS (phosphate buffer solution), and suspending the cells by using a culture solution B to prepare a suspension of 1 × 104-0.5 × 104 for later use.
2) The cell culture plate is coated with 0.1-0.02% agarose for 0.5-1 h.
3) Adding the cell suspension prepared in the step 1) into a culture plate coated with agarose, and performing liquid change treatment every day.
4) And when the embryoid appears after 3-4 days of culture, culturing for 7-10 days until the growth amount of the embryoid reaches the highest peak, collecting the embryoid suspension, standing for precipitation, and suspending by using the culture solution C for later use.
5. Directional differentiation of embryoid bodies into mesenchymal stem cells
1) The prepared embryoid body suspension was inoculated into a matrigel-coated plate.
2) Culturing the embryoid body for 10-14h, and adding culture solution D for induction culture when the embryoid body adheres to the wall.
3) The stem cells are formed after culturing for 5-7 days, and the culture solution E is replaced to continue culturing so as to continue proliferation.
Example 4
The mesenchymal stem cells prepared in the examples 1 to 3 are detected:
1. RT-PCR detects the change condition of each gene in the mesenchymal stem cells, and the primers are as follows:
NANOG:F5'-TGAACCTCAGCTACCCCAG-3',
R 5'-TGGTGGTAGCAACACTAAAG-3'
OCT-4：F5'-CCTCACTTCACTGCATGTA-3'
R 5'-CACTTTTTCTTTTCCCCTAGCT-3'
MSX-1：F5'-CGACAGCCCCGTGCCAGAG-3'
R 5'-GGCGGACCCATCTTCTCCAG-3'
GAPDH:F5'-ATCCCATCATCACCATCTTCC-3'
R 5'-GAGTCCTTCCACGATACCA-3'
experimental procedure
① when the prepared mesenchymal stem cells reach 80%, 0.25% pancreatin digests the mesenchymal stem cells and collects the cells.
② extracting total RNA of mesenchymal stem cells by Trizol method, and performing quality detection and concentration measurement with ultramicro spectrophotometer;
③ reverse transcription of RNA into cDNA using reverse transcription kit;
④ preparing 10 μ L amplification system, reacting at 95 deg.C for 10min, 95 deg.C for 15s, and 75 deg.C for 1min, and circulating 40 times.
The results show that in the mesenchymal stem cells prepared in examples 1-3, the expression levels of the genes NAONG and OCT-4 for inducing the expression of the pluripotent stem cells in the mesenchymal stem cells are hardly expressed, which indicates that the induced mesenchymal stem cells lose the expression of the pluripotent stem cells, and the mesenchymal stem cells belong to mesoderm MSX-1, namely that the expression level of the mesoderm genes in the mesenchymal stem cells is obviously increased, and the characteristics of the mesenchymal stem cells are met. Wherein, the detection result of the mesenchymal stem cells prepared in example 1 is shown in figure 1. The results of stem cell detection obtained in examples 2 to 3 were similar.
2. Flow cytometry surface antigen detection
Experimental procedure
① when the mesenchymal stem cells prepared in each example were fused to 80%, 0.25% of the mesenchymal stem cells were digested with pancreatin and the cells were collected to give a cell number of 2 × 105Each sample;
② cell is resuspended in 1m L DPBS and washed 2 times, 200g, centrifuged for 5 min;
adding 1m L70% precooled alcohol into each sample tube, and resuspending and fixing for 2 h;
③ centrifuging, discarding the fixed solution, adding 200 μ L murine CD29, CD44, CD73, CD90, CD105, CD34, CD45 primary antibody (dilution 1:100), and incubating at room temperature for 30 min;
④ 1m L PBS, and incubating with FITC labeled secondary antibody (dilution of 1:200) for 1h at normal temperature in the dark;
⑤ PBS is washed twice, discarded, and added with a proper amount of PBS to resuspend the cells according to the cell amount, and detected by a flow cytometer.
Flow cytometry analysis shows that the mesenchymal stem cells prepared in examples 1-3 express positive CD29, CD44, CD73, CD90 and CD105, express negative CD34 and CD45, and accord with the expression result of the mesenchymal stem cell surface marker. FIG. 2 shows the result of detecting the mesenchymal stem cell surface marker antigen prepared in example 1. The results of stem cell detection obtained in examples 2 to 3 were similar.
3. Cell viability assay
The viability of the cells prepared in the examples is detected by adopting an MTT method, and the results show that the viability of the mesenchymal stem cells prepared in the examples 1 to 3 is 95%, 62% and 55% in sequence, wherein the viability of the stem cells prepared in the example 1 is obviously higher than that of the stem cells prepared in the examples 2 to 3(p <0.05)
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (5)

1. A culture medium composition for inducing transformation of IPS cells into mesenchymal stem cells, comprising:
the embryoid body induction culture medium consists of a basic culture medium and the following components:
Figure FDA0002387023580000011
the embryoid body culture medium consists of a basic culture medium and the following components:
Figure FDA0002387023580000012
the mesenchymal stem cell induction culture medium consists of a basic culture medium and the following components:
Figure FDA0002387023580000013
the mesenchymal stem cell culture medium consists of a basic culture medium and the following components:
Figure FDA0002387023580000021
2. the culture medium composition according to claim 1, wherein the basal medium is DMEM medium.
3. A method for inducing IPS cells to form embryoid bodies, which is characterized in that the embryoid body induction culture medium of claim 1 is used for inducing the IPS cells, and the induction time is 3-10 days.
4. A method for preparing mesenchymal stem cells from urine cells is characterized by comprising the following steps:
introducing transcription regulatory factors OCT4, SOX2, NANOG, K L F4 and L IN28 into urine cells, and culturing to obtain IPS cells;
inducing IPS cells by using the embryoid body induction culture medium of claim 1, collecting embryoid bodies after 3-10 days, culturing the embryoid body induction culture medium of claim 1 for 10-14h, and inducing for 5-7 days by using the mesenchymal stem cell induction culture medium of claim 1 to obtain mesenchymal stem cells;
proliferating and passaging the mesenchymal stem cells by using the mesenchymal stem cell culture medium of claim 1.
5. The method of claim 4, wherein the urine cells are prepared by: urine was centrifuged, and the pellet was sterilized and cultured in REGM medium containing Primocin to obtain urine cells.
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