CN108148864B - Preparation method of human feeder layer cells capable of supporting growth of human embryonic stem cells - Google Patents

Preparation method of human feeder layer cells capable of supporting growth of human embryonic stem cells Download PDF

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CN108148864B
CN108148864B CN201810030000.4A CN201810030000A CN108148864B CN 108148864 B CN108148864 B CN 108148864B CN 201810030000 A CN201810030000 A CN 201810030000A CN 108148864 B CN108148864 B CN 108148864B
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CN108148864A (en
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邹春林
滕夏虹
王丽惠
许倩倩
孙晓婷
卢奕
张健
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Guangxi Medical University
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Abstract

The invention provides a preparation method of human feeder layer cells capable of supporting the growth of human embryonic stem cells, which comprises the following steps: s1, placing the human bone marrow mesenchymal stem cells in a culture medium for subculture; s2, infecting the human bone marrow mesenchymal stem cells subcultured in S1 with retrovirus particles containing hygromycin resistance genes to obtain human bone marrow mesenchymal stem cells; s3, infecting human mesenchymal stem cells with retrovirus particles containing Wnt3a gene and neomycin resistance gene to obtain TW2R cells; s4, infecting the slow virus particles containing the E-cadherin gene and the puromycin resistance gene into TW2R cells to obtain TWE3R cells. The human feeder layer cell prepared by the invention can stably express the E-cadherin protein, and the proliferation capacity of the TWE3R cell can not be influenced after the E-cadherin gene is stably expressed.

Description

Preparation method of human feeder layer cells capable of supporting growth of human embryonic stem cells
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of human feeder layer cells capable of supporting the growth of human embryonic stem cells.
Background
Human embryonic stem cells have wide application prospects in the aspects of transformation medicine, regenerative medicine and new drug screening, and the existing culture of human embryonic stem cells (hESCs) generally needs mouse embryonic fibroblasts as feeder cells, but the use of the animal-derived materials brings certain biological safety problems for the clinical application in the future. In addition, because only 5 generations of MEFs can be used as feeder layers to culture human embryonic stem cells, and because different batches of MEFs derived from fetal rats have different abilities to maintain the pluripotent growth of human embryonic stem cells, the ability to maintain the pluripotent growth of human embryonic stem cells is different, and the instability is brought to the in vitro long-term culture of human embryonic stem cells. Although TeSR developed recently by STEMCELL corporation, well known in CanadaTM-E8TMThe TeSR-E8 culture medium is a culture medium with highly determined components and without feeder-free cells, is suitable for culturing human embryonic stem cells, and can support the growth of the stem cells without animal proteins, but human serum albumin and human-derived matrix protein are added into the TeSR-E8 culture medium, so that the culture condition is extremely expensive and is not suitable for routine use. Therefore, the research and development of the immortalized human feeder cells which can support the growth of the human embryonic stem cells have important significance for the clinical application of the human embryonic stem cells in the future.
E-cadherin (cadherin E), a member of the subgroup of calcium-dependent adhesion molecules, is a transmembrane protein that is widely distributed in non-neuroepithelial tissues, binds to specific molecules within cells, and participates in homoaffinity cell-to-cell adhesion, embryonic development, and the formation and maintenance of epithelial cell layers in normal tissues. In addition, studies have shown that E-cadherin also plays an important role in maintaining the pluripotency of embryonic stem cells.
Disclosure of Invention
An object of the present invention is to solve the above-mentioned problems and to provide at least the advantages which will be described later.
It is still another object of the present invention to provide a method for preparing a feeder layer cell of human origin capable of supporting the growth of human embryonic stem cells, which can stably transfect an E-cadherin gene to stably express an E-cadherin protein without affecting the proliferation ability of TWE3R cells after stably expressing the E-cadherin gene.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for preparing feeder cells of human origin for supporting the growth of human embryonic stem cells, comprising the steps of:
s1, placing the human bone marrow mesenchymal stem cells in an MSC culture medium for subculture;
s2, infecting the human bone marrow mesenchymal stem cells subcultured in S1 with retrovirus particles containing the hTERT gene and the hygromycin resistance gene, and then adding hygromycin B for screening to obtain the human bone marrow mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene;
s3, infecting the human mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene in S2 with retrovirus particles containing the Wnt3a gene and the neomycin resistance gene, and then adding G418 for screening to obtain TW2R cells containing the hTERT gene, the hygromycin resistance gene, the Wnt3a gene and the neomycin resistance gene;
s4, infecting the slow virus particles containing the E-cadherin gene and the puromycin resistance gene into TW2R cells in S3, and then adding puromycin to carry out screening to obtain TWE3R cells which over-express the E-cadherin, namely the human feeder cells.
Preferably, in the preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the MSC culture medium in S1 contains 0.08-0.1 mL of fetal bovine serum, 0.01-0.015 mL of antimicrobial-antimicrobial, 0.9-1.1 ng of bFGF and the balance of DMEM in each milliliter of culture medium.
Preferably, the method for preparing human feeder cells capable of supporting the growth of human embryonic stem cells, the method for preparing retroviral particles containing the hTERT gene and the hygromycin resistance gene in S2, comprises the following steps:
a1, inoculating 293T cells to a culture dish coated with a polylysine solution in advance, adding high-sugar DMEM containing FBS and no antibiotics, and culturing for 24h, wherein the volume fraction of the FBS is 1%;
a2, adding OPTI-MEM into a container I, then sequentially adding viral plasmids of hTERT RV-Vector, Gag/pol and VSV-G, uniformly mixing and standing; adding OPTI-MEM and Lipofectamine 2000 into the second container, and mixing uniformly; uniformly mixing the raw materials in the first container and the second container;
a3, adding the mixed raw materials in the first container and the second container into a culture dish which is coated with polylysine solution in advance in the A1, uniformly mixing, placing the mixture in an incubator for culturing for 48 hours, collecting cell supernatant containing virus particles, and concentrating to obtain the retrovirus particles containing the hTERT gene and the hygromycin resistance gene;
wherein, hTERT RV-Vector, Gag/pol, VSV-G, OPTI-MEM, DMEM, Lipofectamine 2000
The mass-to-volume ratio of (1) to (6) is 14.9-15.1 mug to (5.8-6.1 mug to) (2.8-3.2 mug to) (1ml to (18-20 ml to) (34-36 mug).
Preferably, the method for preparing human feeder cells capable of supporting the growth of human embryonic stem cells, the method for preparing retroviral particles containing the Wnt3a gene and the neomycin resistance gene in S3, comprises the following steps:
b1, inoculating 293T cells to a culture dish coated with a polylysine solution in advance, adding high-sugar DMEM containing FBS and no antibiotics, and culturing for 24h, wherein the volume fraction of the FBS is 1%;
b2, adding OPTI-MEM into a container III, then sequentially adding viral plasmids of Wnt3a RV-Vector, Gag/pol and VSV-G, uniformly mixing and standing; adding OPTI-MEM and Lipofectamine 2000 into the fourth container, and mixing uniformly; uniformly mixing the raw materials in the container III and the container IV;
b3, adding the mixed raw materials in the third container and the fourth container into a culture dish which is coated with polylysine solution in advance in B1, uniformly mixing, placing the mixture in an incubator for culturing for 48 hours, collecting cell supernatant containing virus particles, and concentrating to obtain the retrovirus particles containing the Wnt3a gene and the neomycin resistance gene;
wherein Wnt3a RV-Vector, Gag/pol, VSV-G, OPTI-MEM, DMEM, Lipofectamine 2000
The mass-to-volume ratio of (1) to (6) is 14.9-15.1 mug to (5.8-6.1 mug to) (2.8-3.2 mug to) (1ml to (18-20 ml to) (34-36 mug).
Preferably, in the preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the lentiviral particles containing the E-cadherin gene and the puromycin resistance gene in S4 are LV6-homo E-cadherin viral particles.
Preferably, the method for preparing human feeder layer cells capable of supporting the growth of human embryonic stem cells comprises subculturing in S1 under CO2Culturing for 60-80 h at the volume concentration of 5-10% and the temperature of 36-37 ℃.
Preferably, the method for preparing human feeder layer cells capable of supporting the growth of human embryonic stem cells comprises the steps of culturing for 48 hours in A3, collecting cell supernatant, adding high-sugar DMEM containing FBS and no antibiotics, the volume of which is equal to that of DMEM in A1, into the mixed raw materials, culturing for 72 hours, collecting cell supernatant again, combining cell supernatants twice, and concentrating to obtain the retrovirus particles containing the hTERT gene and the hygromycin resistance gene, wherein the volume fraction of FBS is 1%.
Preferably, the method for preparing human feeder layer cells capable of supporting the growth of human embryonic stem cells comprises the steps of screening by using 50ug/ml of hygromycin B in S2; the screening was carried out in S3 using 500ug/ml G418; the screening was carried out at S4 using puromycin at 1 ug/ml.
Preferably, the method for preparing human feeder cells capable of supporting the growth of human embryonic stem cells comprises the step of preparing 10 of S15The individual bone marrow mesenchymal stem cells are placed in 1.9-2.1 ml of MSC culture medium for subculture.
Preferably, in the preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, 125-250 mu l of retrovirus particles containing the hTERT gene and the hygromycin resistance gene are infected by 10 in S25In human bone marrow mesenchymal stem cells subcultured in S1; in S3, 125-250 mu l of retrovirus particles containing Wnt3a gene and neomycin resistance gene are infected by 105The human mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene in the S2; 125-250 mu l of gene containing E-cadherin andlentiviral particle infection of puromycin resistance Gene 105TW2R cells in S3.
The invention at least comprises the following beneficial effects:
1. the TWE3R cell prepared by the preparation method can stably transfect the E-cadherin gene so as to stably express the E-cadherin protein, and the proliferation capacity of the TWE3R cell can not be influenced after the E-cadherin gene is transfected; meanwhile, the TWE3R cell can improve the clone formation rate of the H9 human embryonic stem cell, and after the H9 human embryonic stem cell is continuously subcultured on the TWE3R cell for 10 generations, the cell still maintains normal undifferentiated morphology and expresses a specific marker of the human embryonic stem cell.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a schematic representation of the results of immunofluorescence staining of MSC, TW2R, TWN3R and TWE3R cell E-cadherin antibodies and Wnt3a antibodies cells;
FIG. 2 is a schematic representation of the results of Western blot analysis of MSCs, TW2R, TWN3R and TWE3R cells of the E-cadherin antibody, Wnt3a antibody and the reference antibody β -tubulin;
FIG. 3 is a schematic representation of TWN3R and TWE3R cell expansion curves;
FIG. 4 is a graphical representation of the results of a karyotype analysis of TWE 3R;
FIG. 5 is a graph showing the comparison of the clonogenic rates of H9 human embryonic stem cells on TW2R, TWN3R, TWE3R and MEF cells, respectively;
FIG. 6 is a graphical representation of the morphology of H9 human embryonic stem cells following serial passage on TWE3R cells for 10 passages and the results of cellular immunofluorescent staining of human embryonic stem cell markers;
FIG. 7 is a graph showing the results of karyotype analysis of H9 human embryonic stem cells after serial passage for 10 passages on TWE3R cells;
FIG. 8 is a schematic diagram showing the cell immunofluorescence staining results of an in vitro embryoid body formation experiment and a teratoma nodulation experiment in NOD-SCID immunodeficient mice after H9 human embryonic stem cells are continuously passaged on TWE3R cells for 10 generations.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It is to be noted that the experimental methods described in the following embodiments are all conventional methods unless otherwise specified, and the reagents and materials are commercially available unless otherwise specified.
Example 1
A method for preparing human feeder layer cells capable of supporting the growth of human embryonic stem cells comprises the following steps:
s1, placing the human bone marrow mesenchymal stem cells in an MSC culture medium for subculture; the human bone marrow mesenchymal stem cells are MSC cells;
s2, infecting retrovirus particles containing the hTERT gene and the hygromycin (hygromycin B) resistance gene into the human bone marrow mesenchymal stem cells subcultured in S1, and then adding hygromycin B for screening to obtain the human bone marrow mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene;
s3, infecting human mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene in S2 with retrovirus particles containing the Wnt3a gene and the neomycin (neomycin) resistance gene, and then adding G418 for screening to obtain TW2R cells containing the hTERT gene, the hygromycin resistance gene, the Wnt3a gene and the neomycin resistance gene;
s4, infecting the slow virus particles containing the E-cadherin gene and puromycin (puromycin) gene into TW2R cells in S3, and then adding puromycin to carry out screening to obtain TWE3R cells which over-express the E-cadherin, namely the human feeder cells.
The human feeder layer cell can support the preparation method of the human feeder layer cell for the growth of the human embryonic stem cell, and the MSC culture medium in S1 contains 0.08mL of fetal calf serum, 0.01mL of antimicrobial-antimicrobial, 0.9ng of bFGF and the balance of DMEM in each milliliter of culture medium.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the preparation method of the retrovirus particles containing the hTERT gene and the hygromycin resistance gene in S2 comprises the following steps:
a1, inoculating 293T cells to a culture dish coated with a polylysine solution in advance, adding 18ml of high-glucose DMEM containing FBS and no antibiotics, and culturing for 24h, wherein the volume fraction of the FBS is 1%;
a2, adding 1.1ml of OPTI-MEM into a container I, then sequentially adding 14.9 mu G of hTERT RV-Vector, 5.8 mu G of Gag/pol and 2.8 mu G of VSV-G packaging plasmid, uniformly mixing and standing; adding 1.1ml of OPTI-MEM and 34. mu.l of Lipofectamine 2000 into the second container, and uniformly mixing; uniformly mixing the raw materials in the first container and the second container;
a3, adding the mixed raw materials in the first container and the second container into a culture dish which is coated with polylysine solution in advance in the A1, uniformly mixing, placing the mixture in an incubator for culturing for 48 hours, collecting cell supernatant containing virus particles, and concentrating to obtain the retrovirus particles containing the hTERT gene and the hygromycin resistance gene.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the preparation method of the retrovirus particles containing the Wnt3a gene and the neomycin resistance gene in S3 comprises the following steps:
b1, inoculating 293T cells to a culture dish coated with a polylysine solution in advance, adding 18ml of high-glucose DMEM containing FBS and no antibiotics, and culturing for 24h, wherein the volume fraction of the FBS is 1%;
b2, adding 1.1ml of OPTI-MEM into a container III, then sequentially adding 14.9 mu G of hTERT RV-Vector, 5.8 mu G of Gag/pol and 2.8 mu G of VSV-G virus plasmid, uniformly mixing and standing; adding 1.1ml of OPTI-MEM and 34. mu.l of Lipofectamine 2000 into the fourth container, and uniformly mixing; uniformly mixing the raw materials in the container III and the container IV;
and B3, adding the mixed raw materials in the third container and the fourth container into a culture dish which is coated with polylysine solution in advance and mixed uniformly in the B1, placing the mixture in an incubator for culturing for 48 hours, collecting cell supernatant containing virus particles, and concentrating to obtain the retrovirus particles containing the Wnt3a gene and the neomycin resistance gene.
In the preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the lentiviral particles containing the E-cadherin genes and the puromycin resistance genes in the S4 are LV6-homo E-cadherin viral particles. The LV6-homo E-cadherin virus concentrate was synthesized by Shanghai Jima pharmaceutical technology, Inc.
The preparation method of the human feeder layer cell capable of supporting the growth of the human embryonic stem cell comprises the step of subculturing in S1 under CO2The culture was carried out at a volume concentration of 5% and a temperature of 36 ℃ for 60 hours.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells comprises the steps of culturing for 48 hours in A3, collecting cell supernatant, adding high-sugar DMEM containing FBS and no antibiotics, the volume of which is equal to that of DMEM in A1, into the mixed raw materials, culturing for 72 hours, collecting cell supernatant again, combining cell supernatants twice, and concentrating to obtain the retrovirus particles containing the hTERT gene and the hygromycin resistance gene, wherein the volume fraction of the FBS is 1%.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells comprises the steps of screening 50ug/ml hygromycin B in S2; the screening was carried out in S3 using 500ug/ml G418; the screening was carried out at S4 using puromycin at 1 ug/ml.
The preparation method of the human feeder layer cell capable of supporting the growth of the human embryonic stem cell comprises the step of 10 in S15The individual bone marrow mesenchymal stem cells were subcultured in 1.9ml of MSC medium.
The preparation method of the human feeder layer cell capable of supporting the growth of the human embryonic stem cell comprises the step of infecting 125 mu l of retrovirus particles containing the hTERT gene and the hygromycin resistance gene into 10 in S25In human bone marrow mesenchymal stem cells subcultured in S1; in S3, 125. mu.l of retroviral particles containing Wnt3a gene and neomycin resistance gene were infected with 105The human mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene in the S2; in S4, 125. mu.l of a recombinant vector containing the E-cadherin gene andlentiviral particle infection of puromycin resistance Gene 105TW2R cells in S3.
Example 2
A method for preparing human feeder layer cells capable of supporting the growth of human embryonic stem cells comprises the following steps:
s1, placing the human bone marrow mesenchymal stem cells in an MSC culture medium for subculture;
s2, infecting the human bone marrow mesenchymal stem cells subcultured in S1 with retrovirus particles containing the hTERT gene and the hygromycin resistance gene, and then adding hygromycin B for screening to obtain the human bone marrow mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene;
s3, infecting the human mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene in S2 with retrovirus particles containing the Wnt3a gene and the neomycin resistance gene, and then adding G418 for screening to obtain TW2R cells containing the hTERT gene, the hygromycin resistance gene, the Wnt3a gene and the neomycin resistance gene;
s4, infecting the slow virus particles containing the E-cadherin gene and the puromycin resistance gene into TW2R cells in S3, and then adding puromycin to carry out screening to obtain TWE3R cells which over-express the E-cadherin, namely the human feeder cells.
In the preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the MSC culture medium in S1 contains 0.09mL of fetal bovine serum, 0.013mL of biological-antibacterial, 1ng of bFGF and the balance of DMEM in each milliliter of culture medium.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells is characterized in that the preparation method of the retrovirus particles containing the hTERT gene and the hygromycin resistance gene in S2 comprises the following steps:
a1, inoculating 293T cells to a culture dish coated with a polylysine solution in advance, adding 19ml of high-glucose DMEM containing FBS and no antibiotics, and culturing for 24h, wherein the volume fraction of the FBS is 1%;
a2, adding 1.2ml of OPTI-MEM into a container I, then sequentially adding 15 mu G of hTERT RV-Vector, 6 mu G of Gag/pol and 3 mu G of VSV-G virus plasmid, uniformly mixing and standing; adding 1.2ml of OPTI-MEM and 35 ul of Lipofectamine 2000 into the second container, and uniformly mixing; uniformly mixing the raw materials in the first container and the second container;
a3, adding the mixed raw materials in the first container and the second container into a culture dish which is coated with polylysine solution in advance in the A1, uniformly mixing, placing the mixture in an incubator for culturing for 48 hours, collecting cell supernatant containing virus particles, and concentrating to obtain the retrovirus particles containing the hTERT gene and the hygromycin resistance gene.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the preparation method of the retrovirus particles containing the Wnt3a gene and the neomycin resistance gene in S3 comprises the following steps:
b1, inoculating 293T cells to a culture dish coated with a polylysine solution in advance, adding 19ml of high-glucose DMEM containing FBS and no antibiotics, and culturing for 24h, wherein the volume fraction of the FBS is 1%;
b2, adding 1.2ml of OPTI-MEM into a container III, then sequentially adding 15 mu G of Wnt3a RV-Vector, 6 mu G of Gag/pol and 3 mu G of VSV-G virus plasmid, uniformly mixing and standing; adding 1.2ml of OPTI-MEM and 35 ul of Lipofectamine 2000 into the fourth container, and uniformly mixing; uniformly mixing the raw materials in the container III and the container IV;
and B3, adding the mixed raw materials in the third container and the fourth container into a culture dish which is coated with polylysine solution in advance and mixed uniformly in the B1, placing the mixture in an incubator for culturing for 48 hours, collecting cell supernatant containing virus particles, and concentrating to obtain the retrovirus particles containing the Wnt3a gene and the neomycin resistance gene.
In the preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the lentiviral particles containing the E-cadherin genes and the puromycin resistance genes in the S4 are LV6-homo E-cadherin viral particles.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells comprises the step of subculturing in S1With the proviso that2The culture was carried out at 37 ℃ for 70 hours at a volume concentration of 8%.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells comprises the steps of culturing for 48 hours in A3, collecting cell supernatant, adding high-sugar DMEM containing FBS and no antibiotics, the volume of which is equal to that of DMEM in A1, into the mixed raw materials, culturing for 72 hours, collecting cell supernatant again, combining cell supernatants twice, and concentrating to obtain the retrovirus particles containing the hTERT gene and the hygromycin resistance gene, wherein the volume fraction of the FBS is 1%.
In the preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, 50ug/ml of hygromycin B is used for screening in S2; the screening was carried out in S3 using 500ug/ml G418; the screening was carried out at S4 using puromycin at 1 ug/ml.
The preparation method of the human feeder layer cell capable of supporting the growth of the human embryonic stem cell comprises the step of 10 in S15The individual bone marrow mesenchymal stem cells are placed in 2ml of MSC culture medium for subculture.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells comprises the step of infecting 200 mu l of retrovirus particles containing the hTERT gene and the hygromycin resistance gene into 10 in S25In human bone marrow mesenchymal stem cells subcultured in S1; 200. mu.l of retroviral particles containing the Wnt3a gene and the neomycin resistance gene were infected 10 at S35The human mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene in the S2; 200. mu.l of lentiviral particles containing the E-cadherin gene and the puromycin resistance gene were infected 10 at S45TW2R cells in S3.
Example 3
A method for preparing human feeder layer cells capable of supporting the growth of human embryonic stem cells comprises the following steps:
s1, placing the human bone marrow mesenchymal stem cells in an MSC culture medium for subculture;
s2, infecting the human bone marrow mesenchymal stem cells subcultured in S1 with retrovirus particles containing the hTERT gene and the hygromycin resistance gene, and then adding hygromycin B for screening to obtain the human bone marrow mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene; infecting retrovirus particles containing hTERT gene and hygromycin resistance gene into human bone marrow mesenchymal stem cells after subculture in S1;
s3, infecting the human mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene in S2 with retrovirus particles containing the Wnt3a gene and the neomycin resistance gene, and then adding G418 for screening to obtain TW2R cells containing the hTERT gene, the hygromycin resistance gene, the Wnt3a gene and the neomycin resistance gene;
s4, infecting the slow virus particles containing the E-cadherin gene and the puromycin resistance gene into TW2R cells in S3, and then adding puromycin to carry out screening to obtain TWE3R cells which over-express the E-cadherin, namely the human feeder cells.
In the preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the MSC culture medium in S1 contains 0.1mL of fetal calf serum, 0.015mL of Antibiotic-Antibiotic, 1.1ng of bFGF and the balance of DMEM in each milliliter of culture medium.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the preparation method of the retrovirus particles containing the hTERT gene and the hygromycin resistance gene in S2 comprises the following steps:
a1, inoculating 293T cells to a culture dish coated with a polylysine solution in advance, adding 20ml of high-glucose DMEM containing FBS and no antibiotics, and culturing for 24h, wherein the volume fraction of the FBS is 1%;
a2, adding 1.3ml of OPTI-MEM into a container I, then sequentially adding 15.1 mu G of hTERT RV-Vector, 6.1 mu G of Gag/pol and 3.2 mu G of VSV-G virus plasmid, uniformly mixing and standing; adding 1.3ml of OPTI-MEM and 36 mu l of Lipofectamine 2000 into the second container, and uniformly mixing; uniformly mixing the raw materials in the first container and the second container;
a3, adding the mixed raw materials in the first container and the second container into a culture dish which is coated with polylysine solution in advance in the A1, uniformly mixing, placing the mixture in an incubator for culturing for 48 hours, collecting cell supernatant containing virus particles, and concentrating to obtain the retrovirus particles containing the hTERT gene and the hygromycin resistance gene.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the preparation method of the retrovirus particles containing the Wnt3a gene and the neomycin resistance gene in S3 comprises the following steps:
b1, inoculating 293T cells to a culture dish coated with a polylysine solution in advance, adding 20ml of high-glucose DMEM containing FBS and no antibiotics, and culturing for 24h, wherein the volume fraction of the FBS is 1%;
b2, adding 1.3ml of OPTI-MEM into a container III, then sequentially adding 15.1 mu G of Wnt3a RV-Vector, 6.1 mu G of Gag/pol and 3.2 mu G of VSV-G virus plasmid, uniformly mixing and standing; adding 1.3ml of OPTI-MEM and 36 ul of Lipofectamine 2000 into the fourth container, and uniformly mixing; uniformly mixing the raw materials in the container III and the container IV;
and B3, adding the mixed raw materials in the third container and the fourth container into a culture dish which is coated with polylysine solution in advance and mixed uniformly in the B1, placing the mixture in an incubator for culturing for 48 hours, collecting cell supernatant containing virus particles, and concentrating to obtain the retrovirus particles containing the Wnt3a gene and the neomycin resistance gene.
In the preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, the lentiviral particles containing the E-cadherin genes and the puromycin resistance genes in the S4 are LV6-homo E-cadherin viral particles.
The preparation method of the human feeder layer cell capable of supporting the growth of the human embryonic stem cell comprises the step of subculturing in S1 under the condition of CO2The culture was carried out at 37 ℃ for 80 hours at a volume concentration of 10%.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells comprises the steps of culturing for 48 hours in A3, collecting cell supernatant, adding high-sugar DMEM containing FBS and no antibiotics, the volume of which is equal to that of DMEM in A1, into the mixed raw materials, culturing for 72 hours, collecting cell supernatant again, combining cell supernatants twice, and concentrating to obtain the retrovirus particles containing the hTERT gene and the hygromycin resistance gene, wherein the volume fraction of the FBS is 1%.
In the preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells, 50ug/ml of hygromycin B is used for screening in S2; the screening was carried out in S3 using 500ug/ml G418; the screening was carried out at S4 using puromycin at 1 ug/ml.
The preparation method of the human feeder layer cell capable of supporting the growth of the human embryonic stem cell comprises the step of 10 in S15The individual bone marrow mesenchymal stem cells were subcultured in 2.1ml of MSC medium.
The preparation method of the human feeder layer cells capable of supporting the growth of the human embryonic stem cells comprises the step of infecting 250 mu l of retrovirus particles containing the hTERT gene and the hygromycin resistance gene into 10 in S25In human bone marrow mesenchymal stem cells subcultured in S1; s3 mu.l of retroviral particles containing Wnt3a gene and neomycin resistance gene were infected with 10. mu.l5The human mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene in the S2; s4 mu.l of lentiviral particles containing the E-cadherin gene and the puromycin resistance gene were infected with 10. mu.l5TW2R cells in S3.
Comparative example 1
The preparation method of TW2R cells was the same as that of TW2R cells in example 1.
Comparative example 2
TWN3 cell line 3R: LV6NC (unloaded virus, i.e., virus particles containing no target gene but resistance gene) lentivirus concentrate synthesized by Shanghai Jima pharmaceutical technology Co., Ltd is added into TW2R cells in S3 in example 1, and puromycin is added for screening to obtain TWN3R cells over-expressing E-cadherin.
Biological characteristics of MSC, TW2R, TWN3R and TWE3R cells were identified, respectively, and TW2R cells used in the following experiments were obtained by the method of comparative example 1, TWN3R cells were obtained by the method of comparative example 2, and TWE3R cells were obtained by the method of example 1.
1. MSC, TW2R, TWN3R, TWE3R cell immunofluorescence staining
Firstly, MSC, TW2R, TWN3R and TWE3R cells are respectively inoculated in a 24-well plate, and after the cells are attached to the wall, the immunofluorescence staining of the cells is carried out according to the following steps:
1) the medium in the 24-well plate was aspirated, washed once with 1ml of 0.01M PBS, and then fixed with 4% paraformaldehyde for 40 minutes, and then washed three times with 0.01M PBS for 5 minutes each.
2) The reaction was performed with 0.3% TritonX-100 for 20 min.
3) Wash three times with 0.01M PBS for 5 minutes each.
4) 5% Normal blocking was blocked with donkey serum for 1 hour.
5) anti-E-Cadherin antibodies (1:200 dilution) and anti-Wnt 3a antibodies (1:100 dilution) diluted with 0.5% blocking normal donkey serum were added and incubated overnight at 4 ℃.
6) Wash three times with 0.01M PBS for 5 minutes each.
7) Diluted fluorescent secondary antibody was added and incubated at room temperature for 2 hours.
8) Wash three times with 0.01M PBS for 5 minutes each.
9) Cell nucleus counterstaining: adding 250 microliter of DAPI (1ng/ml), incubating for 5 minutes at room temperature in the dark, washing 3 times with PBS, 3 minutes each time;
9) mounting the slide with 10% glycerol-0.01M PBS mounting solution, sealing the periphery of the cover glass with colorless nail polish, and observing and taking pictures under a fluorescence microscope. The results are shown in FIG. 1 (scale: 50 μm), and it can be seen from FIG. 1 that the immunofluorescence staining results of the anti-Wnt 3a antibody show that TW2R, TWN3R and TWE3R stably express Wnt3a protein, while primary MSC does not express Wnt3a protein, and that the immunofluorescence staining results of the anti-E-cadherin antibody show that TWE3R stably expresses E-cadherin protein, while MSC, TW2R and TWN3R do not express E-cadherin protein.
2. Western blot experiments on MSC, TW2R, TWN3R and TWE3R cells
MSC with good growth state, TW2R, TWN3R and TWE3R cells were taken, an appropriate amount of RIPA protein lysate (1ml lysate plus 10. mu.l 100mM PMSF, 10. mu.l 100mM cocktail) was added, the mixture was lysed on ice for 1 hour, the cells were scraped off by cell scraping, the cell lysate was collected and centrifuged at 12000 Xg for 20 minutes at 4 ℃ to obtain a supernatant. Adding 4 Xprotein electrophoresis sample buffer solution into the protein sample according to the proportion of 3:1, mixing uniformly, and then heating and boiling for 5-10 minutes. Proteins were separated by SDS-PAGE and electrotransferred to PVDF membrane. The transfer membrane is firstly sealed by 5% skimmed milk powder at room temperature for 1 hour, then incubated at 4 ℃ overnight under the action of primary antibody (E-cadherin antibody (1:1000), Wnt3a antibody (1:1000), internal reference antibody beta-tubulin (1:1000)), washed by TBST membrane for 3 times, 10 minutes each time, unbound primary antibody is washed off, incubated at room temperature for 60 minutes under the action of anti-rabbit antibody (1:10000) for transfer membrane, washed by TBST membrane for 3 times, 10 minutes each time, after washing unbound secondary antibody, ECL method luminescence, darkroom exposure and development are carried out, and scanning is carried out after washing. As shown in FIG. 2, TW2R, TWN3R and TWE3R cells stably expressed Wnt3a protein after stable transfection of Wnt3a gene, TWE3R cells began to stably express E-cadherin protein after further stable transfection of E-cadherin gene, while primary MSC cells did not express Wnt3a protein and E-cadherin protein.
3. TWN3R and TWE3R cell expansion experiments
Respectively mixing 0.5 × 106TWN3R and TWE3R cells were inoculated into two T75 cell culture flasks, and on day 4 after inoculation, the cells were digested with 0.05% typsin + 0.02% EDTA and trypan blue stained to count the number of viable cells, and then 0.5X 10 cells were added6TWN3R and TWE3R cell suspensions obtained by digestion were inoculated into two new T75 cell culture flasks, and on the 4 th day after inoculation, the cells were digested with 0.05% typsin + 0.02% EDTA and stained with trypan blue to count the number of live cells, and by analogy, serial passages were performed, and cumulative expansion fold of the nth generation cells (number of nth generation cells/number of nth-1 generation cells) x (number of nth-1 generation cells/number of nth-2 generation cells) x … (number of 1 generation cells/number of 0 generation cells).
FIG. 3 is a schematic diagram of TWN3R and TWE3R cell expansion curves, and it can be seen from the graphs that the cell proliferation curves of TWN3R and TWE3R cells in continuous passages show that the stable transfection E-cadherin gene does not affect the proliferation capability of TWE3R cells, and the cell doubling time of TW TWN3R cells and TWE3R cells in the idling control group is not obviously changed in the long-term continuous passage culture process.
4. TWE3R karyotyping
Colchicine (Colcemid) with the concentration of 50 mug/ml is added into TWE3R cells in the logarithmic growth phase, and the cells are continuously cultured for 40-60 min at 37 ℃. Then DPBS washes complete medium, adds 0.05% pancreatin-EDTA to digest for 5min, adds whole serum medium to stop digestion. Centrifuging in a centrifuge tube at 1000r/min for 3 min. Adding 0.075mol/L KCl for hypotonic treatment, fully blowing and beating by using a suction pipe, putting into a water bath cabinet at 37 ℃ for continuous incubation for 10-15 min, then adding a fixing solution (methanol: glacial acetic acid 3:1) for pre-fixing for 10min, centrifuging and removing supernatant. Then adding the stationary liquid, fixing for 30min, then centrifuging and removing the supernatant, then adding the stationary liquid, and fixing at 4 ℃ overnight. Then dropping the fixed cell suspension on borneol, and immediately placing the borneol in an oven at 75 ℃ for 3 hours. G banding the slide is digested in 0.25% pancreatin solution pre-warmed to 37 ℃ for 60s, immediately rinsed 2 times in normal saline, then stained in Giemsa mixture for 5min, rinsed with tap water, and air-dried for microscopic examination. A total of 30 split phases were counted and 3 karyotypes were analyzed. The nuclear karyotype morphology of TWE3R cells is shown in FIG. 4. it can be seen from FIG. 4 that the karyotype analysis results show that TWE3R cells have a normal karyotype.
5. Different feeder layer cells support the culture of human embryonic stem cells
To compare the efficiency of different feeder layer cells in supporting embryonic stem cell growth, the present study compared the efficiency of clonogenic H9 embryonic stem cells on TW2R, TWN3R, TWE3R and MEF feeder layer cells. The specific experimental steps are briefly introduced as follows:
1) plating 250ul of 0.1% gelatin in each well of a 24-well plate, incubating for 1 hour at room temperature, and washing for 1 time by PBS;
2) respectively inoculating mitomycin-treated TW2R, TWN3R, TWE3R and CF1MEF feeder layer cells into a 24-well plate, and culturing overnight under the condition that the cells are just converged;
3) h9 embryonic stem cells cultured in feeder cells were digested with accutase (Invitrogen) to give single cell counts, and the numbers were 1, 10, 100, 1X 103And 1X 104The cells were inoculated into the above 24-well culture plate at a density, and after 7 days of culture, the number of positive clones was counted by alkaline phosphatase staining, and the experiment was repeated 4 times, and then different feeder layer cell counts were comparedEfficiency of embryonic stem cell clone formation. As shown in FIG. 5, it can be seen from FIG. 5 that the clone formation rates of H9 human embryonic stem cells at different densities on TWE3R cells were all significantly higher than those of TW2R, TWN3R and MEF cells, while the clone formation rates between TW2R, TWN3R and MEF cells were not significantly different, and TWE3R cells could support the clone formation of human embryonic stem cells under low density seeding conditions (5 cells/cm 2), while the clone formation of human embryonic stem cells was hardly observed on other feeder cells.
6. Long-term subculture of human embryonic stem cells on TWE3R feeder layer cells
In order to confirm that human embryonic stem cells can be subcultured on the TWE3R feeder layer cells for a long time and maintain the biological characteristics of human embryonic stem cells, H9 human embryonic stem cells were inoculated on a mitomycin-treated human embryonic stem cell culture medium containing TWE3R cells and cultured for 10 successive passages, and then cell immunofluorescent staining, karyotyping, pseudoligand formation and teratoma formation were performed, respectively, and the biological characteristics of human embryonic stem cells serially passaged for 10 passages on TWE3R were analyzed.
1) H9 karyotyping of human embryonic stem cells
The experimental method is the same as the TWE3R karyotype analysis experimental method, the TWE3R cells are changed into H9 human embryonic stem cells in the experimental process, and the nuclear karyotype morphology of the H9 human embryonic stem cells is shown in figure 7.
2) Human embryonic stem cell culture medium composition
Figure BDA0001546209890000141
3) Alkaline Phosphatase (AP) staining of human embryonic stem cells
The human embryonic stem cells cultured for 6 days are washed three times by 0.01M PBS, then fixed for 40 minutes by 4 percent paraformaldehyde at room temperature, and after being washed three times by 0.01M PBS, BCIP/NBT solution (1 piece of BCIP/NBT is dissolved in 10ml deionized water) is added, incubated for 15-20 minutes at room temperature, and washed three times by 0.01M PBS to terminate the reaction.
4) Cellular immunofluorescence staining of human embryonic stem cells
The cell immunofluorescence staining procedure of the human embryonic stem cells is the same as the TWE3R cell immunofluorescence staining experimental method, wherein the antibody dilution ratio is as follows:
Figure BDA0001546209890000142
Figure BDA0001546209890000151
the results of the above experiments are shown in FIG. 6 (with a scale of 100 μm), after the H9 human embryonic stem cells were continuously subcultured on TWE3R cells for 10 generations, the cells still maintained normal undifferentiated morphology, and AP staining and immunofluorescence staining of human embryonic stem cell specific markers TRA-1-60, OCT4, NANOG and SSEA-4 cells all showed positive reactions.
5) Embryoid body formation experiment of human embryonic stem cell
Will be 1 × 106The human embryonic stem cells continuously passaged for 10 generations on TWE3R are inoculated into one hole of a low-adhesion 6-hole culture plate, the liquid is changed every other day, the formed embryoid bodies are inoculated into a 24-hole plate treated by 0.1% gelatin after 8 days, the culture is continued for 2 days, then 4% paraformaldehyde is used for fixation, and immunofluorescence staining is carried out, wherein the antibody dilution ratio is as follows:
Tuj-1(beta-tubulin-III) 1:1000
AFP 1:500
α-SMA 1:500
6) teratoma formation experiment of human embryonic stem cells
Will be 5X 106Human embryonic stem cells serially passaged for 10 passages on TWE3R were digested with collagenase, centrifuged and resuspended in 200ul of 1:1 diluted Matrigel, and then injected into hind limb muscle of NOD-SCID mice, after 4-8 weeks, the teratomas formed were fixed and sectioned and HE stained after paraffin embedding.
The results of the above experiments are shown in FIG. 8 (scale: 100 μm), and it can be seen from FIG. 8 that H9 human embryonic stem cells, after being serially subcultured on TWE3R cells for 10 generations, still have the ability to differentiate into three germ layers, and form embryoid-like bodies in vitro, and that cellular immunofluorescence staining shows that the three germ layer-specific markers Tuj-1(beta-tubulin-III) (ectoderm), AFP (endoderm) and α -SMA (mesoderm) are expressed in the embryoid-like bodies. H9 human embryonic stem cells were serially subcultured on TWE3R cells for 10 passages and injected into NOD-SCID immunodeficient mice for visible teratoma formation, and h.e staining showed visible ectodermal (ectoderm), mesodermal (mesoderm) and endodermal (endoderm) tissue morphologies within the teratoma.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.

Claims (10)

1. A method for preparing human feeder cells capable of supporting the growth of human embryonic stem cells, which comprises the following steps:
s1, placing the human bone marrow mesenchymal stem cells in an MSC culture medium for subculture;
s2, infecting the human bone marrow mesenchymal stem cells subcultured in S1 with retrovirus particles containing the hTERT gene and the hygromycin resistance gene, and then adding hygromycin B for screening to obtain the human bone marrow mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene;
s3, infecting the human mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene in S2 with retrovirus particles containing the Wnt3a gene and the neomycin resistance gene, and then adding G418 for screening to obtain TW2R cells containing the hTERT gene, the hygromycin resistance gene, the Wnt3a gene and the neomycin resistance gene;
s4, infecting the slow virus particles containing the E-cadherin gene and the puromycin resistance gene into TW2R cells in S3, and then adding puromycin to carry out screening to obtain TWE3R cells which over-express the E-cadherin, namely the human feeder cells.
2. The method of claim 1, wherein the MSC culture medium in S1 comprises 0.08-0.1 mL fetal bovine serum, 0.01-0.015 mL biological-antibacterial, 0.9-1.1 ng bFGF, and the balance DMEM per mL culture medium.
3. The method of preparing human feeder cells capable of supporting the growth of human embryonic stem cells according to claim 1, wherein the retroviral particle containing the hTERT gene and the hygromycin resistance gene in S2 is prepared by the steps of:
a1, inoculating 293T cells to a culture dish coated with a polylysine solution in advance, adding high-sugar DMEM containing FBS and no antibiotics, and culturing for 24h, wherein the volume fraction of the FBS is 1%;
a2, adding OPTI-MEM into a container I, then sequentially adding viral plasmids of hTERT RV-Vector, Gag/pol and VSV-G, uniformly mixing and standing; adding OPTI-MEM and Lipofectamine 2000 into the second container, and mixing uniformly; uniformly mixing the raw materials in the first container and the second container;
a3, adding the mixed raw materials in the first container and the second container into a culture dish which is coated with polylysine solution in advance in the A1, uniformly mixing, placing the mixture in an incubator for culturing for 48 hours, collecting cell supernatant containing virus particles, and concentrating to obtain the retrovirus particles containing the hTERT gene and the hygromycin resistance gene;
wherein the mass-volume ratio of the hTERT RV-Vector, the Gag/pol, the VSV-G, OPTI-MEM, the DMEM and the Lipofectamine 2000 is 14.9-15.1 mu g, 5.8-6.1 mu g, 2.8-3.2 mu g, 1ml, 18-20 ml and 34-36 mu l.
4. The method of claim 1, wherein the retroviral particles containing the Wnt3a gene and the neomycin resistance gene in S3 are prepared by the steps of:
b1, inoculating 293T cells to a culture dish coated with a polylysine solution in advance, adding high-sugar DMEM containing FBS and no antibiotics, and culturing for 24h, wherein the volume fraction of the FBS is 1%;
b2, adding OPTI-MEM into a container III, then sequentially adding viral plasmids of Wnt3a RV-Vector, Gag/pol and VSV-G, uniformly mixing and standing; adding OPTI-MEM and Lipofectamine 2000 into the fourth container, and mixing uniformly; uniformly mixing the raw materials in the container III and the container IV;
b3, adding the mixed raw materials in the third container and the fourth container into a culture dish which is coated with polylysine solution in advance in B1, uniformly mixing, placing the mixture in an incubator for culturing for 48 hours, collecting cell supernatant containing virus particles, and concentrating to obtain the retrovirus particles containing the Wnt3a gene and the neomycin resistance gene;
wherein the mass-volume ratio of Wnt3a RV-Vector, Gag/pol, VSV-G, OPTI-MEM, DMEM and Lipofectamine 2000 is 14.9-15.1 mug, 5.8-6.1 mug, 2.8-3.2 mug, 1ml, 18-20 ml and 34-36 mug.
5. A method of producing feeder cells of human origin capable of supporting the growth of human embryonic stem cells as claimed in claim 1 wherein the lentiviral particles comprising an E-cadherin gene and a puromycin resistance gene in S4 are LV6-homo E-cadherin viral particles.
6. The method for preparing feeder layer cells of human origin capable of supporting growth of human embryonic stem cells according to claim 1, wherein the subculture in S1 is carried out under CO2Culturing for 60-80 h at the volume concentration of 5-10% and the temperature of 36-37 ℃.
7. The method of claim 3, wherein the method comprises collecting cell supernatant after culturing in A3 for 48 hours, adding high-sugar DMEM containing FBS and no antibiotics to the mixed material in a volume equal to that of DMEM in A1, culturing for 72 hours, collecting cell supernatant again, combining cell supernatants, and concentrating to obtain retroviral particles containing hTERT gene and hygromycin resistance gene, wherein the volume fraction of FBS is 1%.
8. The method for preparing human feeder layer cells capable of supporting growth of human embryonic stem cells according to claim 1, wherein the screening is performed using hygromycin B at a concentration of 50ug/ml in S2; the screening was carried out in S3 using 500ug/ml G418; the screening was carried out at S4 using puromycin at 1 ug/ml.
9. The method for preparing feeder cells of human origin for supporting growth of human embryonic stem cells according to claim 1, wherein 10 is added in S15The individual bone marrow mesenchymal stem cells are placed in 1.9-2.1 ml of MSC culture medium for subculture.
10. The method for preparing human feeder cells capable of supporting the growth of human embryonic stem cells according to claim 1, wherein 125 to 250. mu.l of retroviral particles containing hTERT gene and hygromycin resistance gene are infected with 10. mu.l of S25In human bone marrow mesenchymal stem cells subcultured in S1; in S3, 125-250 mu l of retrovirus particles containing Wnt3a gene and neomycin resistance gene are infected by 105The human mesenchymal stem cells containing the hTERT gene and the hygromycin resistance gene in the S2; in S4, 125-250 mu l of lentivirus particles containing E-cadherin genes and puromycin resistance genes are infected by 105TW2R cells in S3.
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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
E-Cadherin-Coated Plates Maintain Pluripotent ES Cells without Colony Formation;Masato Nagaoka，et al.;《PLOS ONE》;20061231;第1卷(第1期);e15，摘要 *
E-Cadherin-Expressing Feeder Cells Promote Neural Lineage Restriction of Human Embryonic Stem Cells;Rebecca N. Moore，et al.;《STEM CELLS AND DEVELOPMENT》;20110406;第21卷(第1期);摘要，第31页左栏第2段 *
Efficient Derivation and Genetic Modifications of Human Pluripotent Stem Cells on Engineered Human Feeder Cell Lines;Chunlin Zou，et al.;《STEM CELLS AND DEVELOPMENT》;20120106;第21卷(第12期);摘要，第2299页左栏第2段-右栏第3段 *

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