CN114540282B - Method for obtaining mesenchymal stem cells by induced differentiation of pluripotent stem cells - Google Patents
Method for obtaining mesenchymal stem cells by induced differentiation of pluripotent stem cells Download PDFInfo
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- CN114540282B CN114540282B CN202210165121.6A CN202210165121A CN114540282B CN 114540282 B CN114540282 B CN 114540282B CN 202210165121 A CN202210165121 A CN 202210165121A CN 114540282 B CN114540282 B CN 114540282B
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Abstract
The invention relates to the field of cell biology, in particular to a method for obtaining mesenchymal stem cells by inducing differentiation of pluripotent stem cells. The invention provides a culture medium with reasonable components, so that the induction time is shortened, the process is simple, other animal-derived trophoblast cells are not required to be introduced in the induction process, and the mesenchymal stem cells are finally obtained by means of interaction between the embryoid body induction culture medium and the embryoid body internal cells. The method has general applicability; the mesenchymal stem cells obtained by induction of the scheme of the invention have typical mesenchymal stem cell morphology when observed under a microscope, have fast cell proliferation capability, can be continuously subcultured for more than 20 times, and do not have aging phenomena such as cell volume increase or proliferation slowing; and the expression level of all mesenchymal stem cell surface markers is normal after more than 20 continuous passages, and the mesenchymal stem cell surface markers have osteogenic, adipogenic and chondrogenic differentiation potential.
Description
Technical Field
The invention relates to the field of cell biology, in particular to a method for obtaining mesenchymal stem cells by inducing differentiation of pluripotent stem cells.
Background
Pluripotent stem cells (pluripotent STEM CELLS, PSCs) have a strong potential to self-renew and differentiate into tricermal cells, both properties making pluripotent stem cells an important source of cell therapies for a variety of major diseases. Currently, there are two types of pluripotent stem cells undergoing clinical application development: embryonic stem cells and induced pluripotent stem cells. The two kinds of pluripotent stem cells are utilized to conduct directional induction differentiation of functional cells, so that the specific functional cells with clinical treatment value are obtained, and are research hot spots and difficulties in the field at present.
Mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs) become an important cell type in the fields of regenerative medicine and cell therapy because of their strong self-renewal and multipotency potential, and their clinical safety and effectiveness have been verified in a large number of clinical applications. At present, mesenchymal stem cells mainly come from bone marrow, fat, umbilical cord, cord blood, amniotic membrane, placenta and the like. Tissue-derived mesenchymal stem cells also have a certain difference in proliferation capacity and biological functions due to donor and tissue-derived differences, and therefore, it is necessary to develop other strategies for obtaining a large number of homogeneous MSCs. Induced MESENCHYAL STEM CELLS, iMSCs, which are obtained by directional induced differentiation of human pluripotent stem cells (including embryonic stem cells and induced pluripotent stem cells), can provide a stable seed cell for cell therapy. At present, a large number of documents report on the utilization of the strategy to obtain mesenchymal stem cells, and iMSCs has stronger proliferation capacity, stronger immune regulation capacity, neuroprotection capacity and lower tumorigenicity compared with tissue-derived MSCs.
At present, the technical method for qualitatively inducing and obtaining iMSCs by utilizing pluripotent stem cells has some technical defects, such as long induction time and complicated process, the induction process needs animal-derived trophoblast cells, the induction process needs flow separation and purification iMSCs, the obtained iMSCs has the problems of slow proliferation, abnormal immunophenotype or abnormal three-line differentiation potential, and the like, and the technical defects obviously limit the clinical application advantages of iMSCs and tissue-derived MSCs.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a culture medium with reasonable composition, so that the induction time is shortened, the process is simple, and the induction process does not need to introduce animal-derived trophoblast cells.
The invention provides a culture medium comprising basal medium, 2mmol/L glutamine (Glutamine), 1x unnecessary amino acid (NEAA), 1[ mu ] g/ml to 100 [ mu ] g/ml Sodium ascorbate (Na Ascorbic acid), 0.1 to 2.0vol% lipid mixture (CD lipid concentrate), 0.1 to 10mg/ml recombinant human serum albumin (Recombinant Human serum Albumin), 1ng to 10ng/ml Sodium selenite (Sodium selenium), 1[ mu ] g to 200 [ mu ] g/ml whole iron transferrin (Holo-transferrin), 0.1 [ mu ] g to 20 [ mu ] g/ml Insulin (Inulin), 0.1 [ mu ] mol to 50 [ mu ] mol/L ethanolamine (Ethanolamine), 1[ mu ] mol to 200 [ mu ] mol/L monothioglycerol (Monothioglycerol) and 0.01 to 0.1wt% polyvinyl alcohol (Polyvinylaohol).
In some embodiments, the medium consists of basal medium, 2mmol/L glutamine, 1 Xunnecessary amino acids, 1. Mu.g/ml ascorbic acid sodium salt, 0.1vol% lipid mixture, 0.1mg/ml recombinant human serum albumin, 1ng/ml sodium selenite, 1. Mu.g/ml whole iron transferrin, 0.1. Mu.g/ml insulin, 0.1. Mu.mol/L ethanolamine, 1. Mu.mol/L monothioglycerol, and 0.01wt% polyvinyl alcohol.
In other embodiments, the medium consists of basal medium, 2mmol/L glutamine, 1X optional amino acid, 50. Mu.g/ml sodium ascorbate, 1.0vol% lipid mixture, 1mg/ml recombinant human serum albumin, 5ng/ml sodium selenite, 10. Mu.g/ml whole iron transferrin, 10. Mu.g/ml insulin, 20. Mu.mol/L ethanolamine, 200. Mu.mol/L monothioglycerol, and 0.05wt% polyvinyl alcohol.
In other embodiments, the medium consists of basal medium, 2mmol/L glutamine, 1 Xunnecessary amino acids, 100. Mu.g/ml sodium ascorbate, 2.0vol% lipid mixture, 10mg/ml recombinant human serum albumin, 10ng/ml sodium selenite, 200. Mu.g/ml whole iron transferrin, 20. Mu.g/ml insulin, 50. Mu.mol/L ethanolamine, 200. Mu.mol/L monothioglycerol, and 0.1wt% polyvinyl alcohol.
In the culture medium provided by the invention, the basal culture medium comprises a DMEM/F12 culture medium.
The culture medium provided by the invention further comprises an antibiotic, wherein the antibiotic is at least one selected from streptomycin, chloramphenicol, kanamycin, tetracycline, penicillin, methicillin and nalidixic acid.
In some embodiments, streptomycin and penicillin are also included in the medium.
The invention provides application of the culture medium in inducing differentiation of pluripotent stem cells to embryoid bodies.
In the present invention, the pluripotent stem cells are human pluripotent stem cells.
The invention also provides a method for inducing the differentiation of the pluripotent stem cells to the embryoid bodies, and the embryoid bodies are obtained by using the culture medium to induce the pluripotent stem cells.
The invention provides a method for obtaining mesenchymal stem cells by induced differentiation of pluripotent stem cells, comprising the following steps:
step 1: culturing pluripotent stem cells;
Step 2: inducing pluripotent stem cells by using the culture medium of the invention to obtain embryoid bodies;
Step 3: the embryoid body is induced to obtain mesenchymal stem cells.
In the method, the culture medium in the step 1 is mTESR1 culture medium, and the culture is carried out until the clone confluence reaches 80% -90%.
In step 1, the inoculation ratio of the cultured pluripotent stem cells is 5:1.
In the method of the present invention, the conditions for induction in step 2 include culturing for 10 days, changing fresh medium after culturing for 24 hours, and changing fresh medium every other day thereafter. In step 2, the inoculation ratio of the induction is 1:1.
In the method of the present invention, the conditions for induction in step 3 include culturing for 7 days using MSC complete medium. In the step 3, embryoid bodies are inoculated according to the ratio of 1:2 in the induction.
In some examples, the pluripotent stem cells of the invention are human pluripotent stem cells.
In some embodiments, the mesenchymal stem cells are inoculated on a common cell culture plate for culture according to the density of 4×10 4cells/cm2, the culture medium is MSC complete culture medium, and subculture is carried out when the cell confluency is 80% -90%.
The mesenchymal stem cells provided by the invention can be continuously passaged for 20 times.
The culture medium provided by the invention comprises basal culture medium, glutamine (Glutamine), 1 x non-essential amino acid (NEAA), sodium ascorbate (Na Ascorbic acid), lipid mixture (CD lipid concentrate), recombinant human serum albumin (Recombinant Human serum Albumin), sodium selenite (Sodium selenium), total iron transferrin (Holo-transferrin), insulin (Insulin), ethanolamine (Ethanolamine), monothioglycerol (Monothioglycerol) and polyvinyl alcohol (Polyvinyllankohol). According to the invention, three hESCs (H1/HUES-9/H9)/two hiPSCs (PBMC-iPS and FB-iPS) are adopted to carry out a mesenchymal stem cell induction experiment, and as a result, 5 cells are successfully induced to obtain mesenchymal stem cells, so that the method has universal applicability; the induced mesenchymal stem cells obtained by the invention have typical mesenchymal stem cell morphology when observed under a microscope, have fast cell proliferation capability, can be continuously subcultured for more than 20 times, and do not have aging phenomena such as cell volume increase or proliferation slowing; the iMSCs obtained by the invention expresses the mesenchymal stem cell surface marker, and the mesenchymal stem cell surface marker expression level is normal after continuous passage for more than 20 times; and iMSCs obtained by the invention has the differentiation potential of osteogenesis, adipogenesis and chondrogenesis. The two-dimensional induction method is also suitable for iMSCs obtained from different hESCs, but the obtained iMSCs has limited passage capacity (3-5 generations), and part of MSC surface markers have lower expression level, and the osteogenic, adipogenic and chondrogenic differentiation capacity of cells is weaker than that of the examples. Compared with the prior art, the induction method disclosed by the invention has the advantages of low technical difficulty, low requirement on initial cell strains, strong universality and low cost, is a simple, convenient and quick technical method for obtaining the mesenchymal stem cells through the pluripotent stem cells, and lays an important foundation for industrialization of the induced mesenchymal stem cells.
Drawings
For a clearer description of embodiments of the invention or of the solutions of the prior art, the drawings that are needed in the description of the embodiments or of the prior art will be briefly described, it being obvious that the drawings in the description below are some embodiments of the invention, and that, without the inventive effort, other drawings can be obtained from them to those skilled in the art:
FIG. 1 shows iMSCs morphological observations of different generations;
FIG. 2 shows iMSCs p flow assay results showing that all surface marker expression levels meet MSC assay standard requirements;
FIG. 3 shows iMSCs that the results of the three-line differentiation potential identification show that iMSCs has osteogenic, adipogenic and chondrogenic differentiation potential;
FIG. 4 shows iMSCs flow assay results obtained by two-dimensional induction, iMSCs p flow assay results show that CD105 expression levels are low, negative: HLADR, CD34, CD45, CD11b, CD19 antibody mixture detection results;
FIG. 5 shows identification of three-lineage differentiation potential of iMSC by two-dimensional induction method, and iMSCs shows that iMSCs obtained by two-dimensional induction method has osteogenic, adipogenic and chondrogenic differentiation potential.
Detailed Description
The invention provides a method for obtaining mesenchymal stem cells by induced differentiation of pluripotent stem cells, and the technical parameters can be properly improved by a person skilled in the art by referring to the content of the invention. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
NEAA of the present invention was commercially available (Gibco, cat. No.: 11140050) and was added at the recommended concentration (stock solution was 100X, and use concentration was 1%).
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention.
Example 1
1.1 Human pluripotent Stem cell culture
HESCs (H1/HUES-9/H9)/hiPSCs (PBMC-iPS, FB-iPS) are cultured on a Matrigel/Vitronnectin coated culture plate, the culture medium is mTESR1, and the culture medium can be used for mesenchymal stem cell induced differentiation experiments when the cloning confluence reaches 80% -90%; the method comprises the following steps:
(1) Absorbing mTESR1, washing with DMEM/F12 for 1 time;
(2) 0.5ml dispase (1 mg/ml)/well (6 well plate), digestion for 5min;
(3) When the edge of the cell clone was observed under a mirror to be shiny and curled, the cell clone was washed with DMEM/F12 (1 ml/well) for 2 times;
(4) After the DMEM/F12 is sucked and removed, 1 mlmTeSR/hole is added, a 1ml pipette tip is used for dividing the cell clone into cell clusters with uniform size by adopting a mechanical dividing method, and then the cell clusters are scraped off from the bottom of a culture dish gently;
(5) After most of the cell mass is scraped off, observing the size and uniformity of the cell mass under a lens, and if the clone is bigger, blowing the cell mass with a 1ml liquid-transferring gun for 2-3 times to finally obtain the relatively uniform cell mass with moderate size;
(6) Transfer the pellet to a 15ml centrifuge tube and centrifuge at room temperature (1000 rpm,5 min);
(7) The supernatant was discarded, the cell mass was resuspended in embryoid body induction medium (medium formulation see Table 1), added to a low adhesion cell culture plate (inoculated at a ratio of 3-6:1), and transferred to a cell incubator for cultivation at 37℃under 5% CO 2 saturation humidity.
1.2 Embryoid body formation and intermediate cell Induction
After culturing the hESCs/hiPSCs in embryoid body induction medium for 24h, collecting the inducer, centrifuging at low speed (300 rpm,3 min), carefully discarding the supernatant, re-suspending with embryoid body induction medium, transferring embryoid bodies onto new low adhesion cell culture plates (1:1) by Pasteur pipette, culturing for 10 days in total.
TABLE 1 embryoid body induction medium formulation
1.3 Mesenchymal Stem cell Induction
The embryoid body induced for 10 days is inoculated on a common cell culture plate according to the proportion of 1:2, and is replaced by a complete culture medium of MSC, and the culture is carried out for 7 days by changing the liquid every other day.
1.4 Mesenchymal Stem cell purification and expansion culture
(1) Digesting and inducing cell cultures for 7 days by using pancreatin substitutes, counting by using trypan blue staining, detecting the survival rate of the cells, inoculating the cells to a common cell culture plate according to the density of 4 multiplied by 10 4cells/cm2 according to the result, continuously culturing, marking the cells as P0, culturing the cells to reach the cell confluency of 80 to 90 percent by using a culture medium which is MSC complete culture medium, and carrying out subculture;
(2) From P1 to P3, the cell seeding density was 2X 10 4cells/cm2, and from the generation P4, the cell seeding density was changed to 1X 10 4cells/cm2. The MSC surface markers were detected every 2-3 times from P3. And selecting representative generation induced mesenchymal stem cells for bone formation, fat formation and cartilage formation differentiation potential detection.
1.5 Experimental results
(1) General applicability of the Induction method
The scheme adopts three hESCs (H1/HUES-9/H9)/two hiPSCs (PBMC-iPS and FB-iPS) to carry out mesenchymal stem cell induction experiments, and 5 cells are successfully induced to obtain mesenchymal stem cells, so that the method has general applicability;
(2) Basic biological characteristics of induced mesenchymal stem cells
The induced mesenchymal stem cells (MESENSHYMAL STEM CELLS, iMSCs) obtained by the scheme have typical mesenchymal stem cell morphology when observed under a microscope, have fast cell proliferation capability, can be continuously subcultured for more than 20 times, and do not have aging phenomena such as cell volume increase or proliferation slowing (figure 1).
(3) MSC surface marker detection:
The iMSCs obtained in the scheme expresses the mesenchymal stem cell surface markers, and the mesenchymal stem cell surface markers are continuously passaged for more than 20 times, and the expression level of all the mesenchymal stem cell surface markers is normal (table 2 and figure 2).
TABLE 2 Logistics detection results of different generations of iMSC surface markers
(4) Differentiation potential identification
Experimental results show that iMSCs obtained in the scheme has osteogenic, adipogenic and chondrogenic differentiation potential (see figure 3).
Comparative example:
hESCs (H1/HUES-9/H9)/hiPSCs (PBMC-iPS, FB-iPS) are cultured on a Matrigel/Vitronnectin coated culture plate, and when the culture medium is mTESR1 and the cloning confluency reaches 80-90%, the method can be used for mesenchymal stem cell induced differentiation experiments, and comprises the following steps:
(1) Absorbing mTESR1, washing with DMEM/F12 for 1 time;
(2) 0.5ml dispase (1 mg/ml)/well (6 well plate), digestion for 5min;
(3) When the edge of the cell clone was observed under a mirror to be shiny and curled, the cell clone was washed with DMEM/F12 (1 ml/well) for 2 times;
(4) After the DMEM/F12 is sucked and removed, 1ml mTESR 1/hole is added, a 1ml pipette tip is used for dividing the cell clone into cell clusters with uniform size by adopting a mechanical dividing method, and then the cell clusters are scraped off from the bottom of a culture dish gently;
(5) After most of the cell mass is scraped off, observing the size and uniformity of the cell mass under a lens, and if the clone is bigger, blowing the cell mass with a 1ml liquid-transferring gun for 2-3 times to finally obtain the relatively uniform cell mass with moderate size;
(6) Transfer the pellet to a 15ml centrifuge tube and centrifuge at room temperature (1000 rpm,5 min);
(7) Discarding the supernatant, culturing for 3-4 days by mTESR1, and changing liquid every day;
(8) When hESCs were grown to a confluence of 70-80%, the mesenchymal stem cells were replaced with a medium for induction (KOSR medium (DMEM/F12+20% Knock Out serum replacement (KSR) +1mM glutamine+10mM NEAA) +SB431542 (10. Mu.M)), and the medium was changed every day, and the cells were subjected to liquid change every day, (9) to 10 days, and the cell culture was digested into single-cell suspension with a pancreatin replacement, and the mesenchymal stem cells were purified and expanded according to the method of 1.4 in example 1, and the method for induction was performed according to the method of 1.5 in example 1, and the test of the passaging capacity of iMSCs, the detection of cell surface markers, and the identification of differentiation potential were performed.
The results show that the two-dimensional induction method is equally applicable to iMSCs obtained from different hESCs induction, but the obtained iMSCs has limited passage capacity (3-5 generations), and the expression level of partial MSC surface markers is lower (figure 4), and the osteogenic, adipogenic and chondrogenic differentiation capacity of the cells is weak compared with the examples (figure 5).
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (7)
1. A culture medium, characterized in that the culture medium consists of basic culture medium DMEM/F12,2mmol/L glutamine, 1 x nonessential amino acids, 50 μg/ml ascorbic acid sodium salt, 1wt% lipid mixture, 1mg/ml recombinant human serum albumin, 5ng/ml sodium selenite, 10 μg/ml whole iron transferrin, 10 μg/ml insulin, 20 μmol/L ethanolamine, 200 μmol/L monothioglycerol, 0.05wt% polyvinyl alcohol and 1wt% penicillin/streptomycin.
2. Use of the culture medium of claim 1 for inducing differentiation of pluripotent stem cells into embryoid bodies.
3. A method for inducing differentiation of pluripotent stem cells into embryoid bodies, comprising inducing pluripotent stem cells using the medium according to claim 1, and obtaining embryoid bodies.
4. A method for obtaining mesenchymal stem cells by induced differentiation of pluripotent stem cells is characterized in that,
Step 1: culturing pluripotent stem cells;
Step 2: inducing pluripotent stem cells using the medium of claim 1 to obtain embryoid bodies;
Step 3: the embryoid body is induced to obtain mesenchymal stem cells.
5. The method according to claim 4, wherein the medium for the culture in step 1 is mTESR1 medium, and the culture is carried out until the clone confluency reaches 80% -90%.
6. The method of claim 4, wherein the conditions of induction in step 2 comprise changing fresh medium after 24 hours of incubation, changing fresh medium every other day thereafter, and co-incubating for 10 days.
7. The method of claim 4, wherein the conditions of induction in step 3 comprise culturing for 7 days using MSC complete medium.
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| CN107858328A (en) * | 2017-11-15 | 2018-03-30 | 广州赛隽生物科技有限公司 | A kind of neural crest pedigree mesenchymal cell and its method of inducing differentiation from multipotential stem cell |
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