Patent Application
20130040390
The present invention relates to the field of a cell culture medium, and more particularly to a culture medium supplement, which can be used to culture induced pluripotent stem (iPS) cells or mammalian cells.
In 2008, Japanese scientist Yamanaka and his coworkers successfully reprogrammed mouse fibroblasts into embryonic stem (ES) cells with the four transcription factors, Oct4, Kif4, Sox2, and c-Myc, and this technology is referred to as iPS cell technology. Since then, fibroblasts derived from human and other species such as rat, monkey and pig, are also successfully reprogrammed, and thus this technology is considered as an important breakthrough in regeneration medicine. At present, almost all the culture systems used in reprogramming of mice contain serum. Use of the serum culture system has the following disadvantages. 1. The reprogramming process is slow. Serum is confirmed to contain some substances inhibiting reprogramming, for example, TGF-b family growth factors. In addition, other unknown ingredients in serum may also very likely delay the reprogramming process. These confirmed or unconfirmed substances make the reprogramming efficiency extremely low. 2. The serum stability is different from batch to batch. The preparation progression of serum determines that the ingredients of each batch of serum are different, which very likely leads to the decrease of the experimental repeatability. 3. The uncertainty of the ingredients in serum sets obstacles in study of the reprogramming mechanism and in drug screening. Therefore, the ingredients of serum are complex, and various pathways favorable to or unfavorable to the iPS process may be activated, which causes great background noises to the study of the iPS cell mechanism, and decrease the sensitivity of the drug screening.
Researches suggest that the invention of knockout serum replacement (KOSR) serum-free culture medium is a great advance in cell culture. The system is completely free of serum, and most of the ingredients in the system are clear, so that the experimental repeatability is much improved. In contrast to serum, the system almost contains no differentiation factors, which is better for the maintaining of the self-renewal of the stem cells. Furthermore, a mixture of the system with serum exhibits a higher reprogramming efficiency than that exhibited when serum is used alone.
However, the ingredients in the system are still not absolutely clear, for example, the bovine serum globulin complex (Albumax) contains many unknown lipids, and the effect of these ingredients on reprogramming is still unknown. Moreover, KOSR cannot support the growth of the cells in the early stage of the reprogramming. In addition, in induction of mouse iPS cells, the reprogramming efficiency is not high enough even if KOSR is mixed with serum. Therefore, a chemically defined high-efficiency reprogramming system free of serum is very important for the study of the iPS cell mechanism, the drug screening, and the clinical use of iPS cells.
In order to overcome the technical problems above, the present invention provides a chemically defined culture system, by which the iPS cells can be obtained with high efficiency. The culture system can maintain the growth and proliferation of the stem cells in absence of feeder cells, accelerate the iPS cell progression, and improve the iPS efficiency.
In order to achieve the above objectives, the following technical solution is employed.
The culture medium supplement of the present invention includes vitamin C and a glycogen synthase kinase-3 inhibitor.
The culture medium supplement of the present invention may further include, in addition to vitamin C and the glycogen synthase kinase-3 inhibitor, vitamin B12, insulin, a receptor tyrosine kinase, and an anti-oxidant.
In order to achieve the above objectives, the following technical solution is employed.
The vitamin C includes ascorbic acid and a derivative thereof, for example, sodium ascorbate salt, and a stable form of the vitamin C, i.e. 2-phospho-ascorbic acid. A preferred form is 2-phospho-ascorbic acid. A working concentration of vitamin C is, but not limited to, 0-100 μg/ml, and is preferably 50 μg/ml. A working concentration of vitamin B12 is, but not limited to 0-2.8 μg/ml, and is preferably 1.4 μg/ml.
The insulin includes extracted and artificially synthesized recombinant insulin derived from various sources and having biological activity. The recombinant human insulin (SIGMA Inc.) is preferred, and a working concentration is, but not limited to, 0-50 μg/ml, and is preferably 20 μg/ml.
The glycogen synthase kinase-3 inhibitor includes at least one of Li+, Chir99021, BIO, and SB216763. Li+ is preferably in the form of LiCl, and a working concentration is without limitation in the range of 0-10 μg/ml, and is preferably 5 mmol/ml. The glycogen synthase kinase is a class of serine/threonine phosphorylase kinase, and is involved in regulation of many signaling pathways. An inhibitor thereof is preferably CHIR99021, and a working concentration is without limitation in the range of 0-12 μmol/ml, and is preferably 3 μmol/ml.
The receptor tyrosine kinase includes at least one of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and hepatocyte growth factor (HGF). The bFGF is preferred, and a working concentration is in the range of 0-100 ng/ml, and is preferably 5 ng/ml.
The anti-oxidant includes at least one of thiamine, superoxide dismutase (SOD), catalase, reduced glutathione, vitamin E, acetylated vitamin E, linoleic acid, Linolenic acid, and sodium selenite. A working concentration of thiamine is without limitation in the range of 0-36 μg/ml, and is preferably 9 μg/ml; a working concentration of superoxide dismutase is without limitation in the range of 0-10 μg/ml, and is preferably 2.5 μg/ml. A working concentration of the reduced glutathione is without limitation in the range of 0-6 μg/ml, and is preferably 1.5 μg/ml. A working concentration of vitamin E is without limitation in the range of 0-16 μg/ml, and is preferably 1 μg/ml. A working concentration of the acetylated vitamin E is without limitation in the range of 0-16 μg/ml, and is preferably 1 μg/ml. A working concentration of linoleic acid is without limitation in the range of 0-4 μg/ml, and is preferably 1 μg/ml. A working concentration of ethanolamine is without limitation in the range of 0-16 μg/ml, and is preferably 1 μg/ml.
The culture medium supplement of the present invention may be a mixture of the above two culture medium supplements respectively with a serum replacement cell growth promoter, in which the serum replacement cell growth promoter includes at least one of albumin hydrolyzate, transferrin, triiodothyronine, adrenalone, lipoic acid, ethanolamine, progesterone, putrescine, and vitamin A.
The albumin hydrolyzate is a hydrolyzed product of albumin, and is composed of amino acids and polypeptides, the specific ingredients are clear, and a use concentration is without limitation in the range of 0-10 mg/ml, and is preferably 1 mg/ml.
The transferrin includes a transferrin with and without iron ions, and is preferably a transferrin with iron ions, and a use concentration is without limitation in the range of 0-200 μg/ml, and is preferably 100 μg/ml.
A working concentration of the lipoic acid is without limitation in the range of 0-3.2 μg/ml, and is preferably 0.2 μg/ml.
A working concentration of the vitamin A is without limitation in the range of 0-1.6 μg/ml, and is preferably 0.1 μg/ml.
The present invention further provides a complete culture medium, which is formed by one or more of a basal culture medium, serum, and a serum replacement supplement, and the above several culture medium supplements; or formed by a basal culture medium and the above several culture medium supplements. Meanwhile, ingredients are as configured in Table 1 and added to a basal culture medium (Dulbecco's Modified Eagle's Medium, DMEM) to form a chemically defined complete culture medium iCD1. Table 1 shows ingredients of an optimized culture medium supplement and concentrations thereof.
The present invention further provides a complete culture medium for iPS cells, which is formed by one or more of a basal culture medium, serum, and a serum replacement supplement, and the above two culture medium supplements.
The basal culture medium includes, but is not limited to, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), F-10, F-12, RPMI 1640, GMEM (Glasgow's Minimal Essential Medium), αMEM (αMinimal Essential Medium), Iscove's Modified Dulbecco's Medium and M199. DMEM is preferred.
The serum replacement supplement includes, but is not limited to, KOSR, N2, B27, and ITS (Insulin-Transferrin-Selenium Supplement).
Furthermore, the culture medium supplements are configured based on the ingredients shown in Table 1, and added to a basal culture medium DMEM, to form a complete culture medium iCD1 with clear chemical ingredients. Table 1 shows ingredients of an optimized culture medium supplement and concentrations thereof. Table 2 shows ingredients of an optimized complete culture medium iCD1 and concentrations thereof.
The culture system of the present invention is a chemically defined serum-free culture system, which can be used to obtain iPS cells from somatic cells with high efficiency, maintain the growth and proliferation of the fibroblasts and the stem cells in absence of feeder cells, accelerate the iPS progression, and improve the iPS efficiency. Moreover, the culture system of the present invention is useful in eukaryotic cell culture.
In order to make the present invention more comprehensible, the present invention is further described with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention, but not limit the scope thereof.
It should be noted that in the examples below, unless specifically stated otherwise, the terms and ingredients of the culture medium are as described below.
The basal culture medium is an artificially prepared culture containing carbohydrates, amino acids, inorganic salts, vitamins, lipids, and other nutrients required for cell growth. The basal culture medium in the present invention includes, but is not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), F-10, F-12, RPMI 1640, Glasgow's Minimal Essential Medium (GMEM), αMinimal Essential Medium (αMEM), Iscove's Modified Dulbecco's Medium, and M199, with the preferred basal culture medium being high sugar DMEM basal culture medium.
The fetal bovine serum is a culture supporting cell growth isolated and extracted from fetal bovine blood, which contains abundant nutrients and growth factors and can support the growth of a variety of types of cells, but the specific ingredients of which are unknown. Therefore, the fetal bovine serum has the disadvantages such as unclear ingredients, high batch difference, and potential presence of animal pathogens.
KOSR is a class of commercialized culture supplements replacing serum, and generally has clear ingredients. The KOSR described herein includes, but is not limited to, serum replacement supplements such as KSR (a commercialized serum-containing culture supplement), N2 (a commercialized serum replacement supplement, and ingredients thereof include insulin, transferrin, progesterone, putrescine, and sodium selenite), and B27 (a serum free culture supplement used to culture nerve cells).
Embryonic stem cells are shortly referred to as ES or EK cells. The ES cells are a class of cells isolated from an early embryo (before gastrula stage) or from primordial gonad, which have properties of unlimited proliferation in in-vitro culture, self-renewal, and multipotent differentiation. In-vitro culture of the mouse ES cells requires the support of the feeder cells and the mouse leukemia inhibitory factor (LIF) to maintain the pluripotent state. It is found through researches that in an in-vitro culture process, use of a glycogen synthase kinase inhibitor and a mitogen activated protein kinase inhibitor in combination can inhibit the differentiation of the ES cells, and maintain the pluripotent state of the ES cells in absence of feeder cells and mouse LIF.
Glycogen synthase kinase-3 (GSK-3), which is a multifunctional serine/threonine protein kinase, is an important component in multiple intracellular signaling pathways, and is involved in a variety of physiological processes such as intracellular glucose metabolism, cell proliferation, and cell differentiation and apoptosis. The GSK inhibitor mainly includes, for example, CHIR99021, BIO, and SB216763. CHIR99021 is preferred, and the structure thereof is:
iPS cells are an abbreviation for induced pluripotent stem cells, and are a pluripotent stem cell state restored by treating differentiated cells with reprogramming factors. At present, it is confirmed through a tetraploid blastocyst injection technology that the iPS cells can produce a complete mouse, suggesting that the iPS cells are pluripotent.
The reprogramming factors refer to factors able to restore differentiated cells to a pluripotent state, and are mostly nuclear transcription factors. The currently reported transcription factors for reprogramming include, for example, SOX2, OCT3, KIF4, NONOG, LIN28, c-myc, 1 in28, esrrb, and tbx3. The transcription factors as described herein include, but are not limited to, the above transcription factors.
The ingredients of a feeder medium include a high-sugar basal culture medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Hyclone), 2 mM glutamine, and non-essential amino acids (NEAAs).
The ingredients of a serum-containing ES cell culture medium include a high-sugar basal culture medium (DMEM, a basal culture medium named with a first letter of the inventor), 15% fetal bovine serum FBS (GIBCO), 2 mM glutamine, non-essential amino acids (NEAAs), penicillin/streptomycin, beta-mercaptoethanol, and sodium pyruvate.
mES is a classical stem cell culture medium, and has the following ingredients: high-sugar DMEM culture medium supplemented with 15% fetal bovine serum, non-essential amino acids, glutamine, penicillin/streptomycin, beta-mercaptoethanol, pyruvic acid, and leukocyte inhibitory factor (a growth factor maintaining the pluripotent state of mice).
KSR serum-free culture medium: KSR is an abbreviation for Knockout Serum Replace, and is a commercialized serum replacement stem cell culture supplement. The KSR complete culture medium used in examples of the present invention includes two types, one is used in an induction process of the iPS cells, and ingredients thereof include a high-sugar basal culture medium (DMEM), 10% KSR supplement, 2 mM glutamine, non-essential amino acids (NEAAs), penicillin/streptomycin, beta-mercaptoethanol, and sodium pyruvate. The other one is a complete KSR serum-free culture medium used to culture stem cells or iPS clones, and ingredients thereof include KNOCKOUT DMED (a basal culture medium with optimized osmotic pressure and suitable for stem cell culture), 15% KSR supplement, 2 mM glutamine, non-essential amino acids (NEAAs), penicillin/streptomycin, and Beta-mercaptoethanol. All culture media for iPS process and clone are supplemented with mouse leukocyte inhibitory factor LIF (millipore, trade name ESGRO, a growth factor inhibiting differentiation of mouse stem cells at a finally concentration of 1000 U/ml.
KSR-BN is the KSR complete culture medium used in the induction process of the iPS cells and supplemented with bFGF and N2 (a commercialized serum replacement supplement having ingredients including insulin, transferrin, progesterone, putrescine, and sodium selenite).
Basal3.0 complete culture medium refers to one of the culture media described in the present invention, and is specifically formed by a high-sugar basal culture medium (DMEM) supplemented with vitamin C, insulin, lithium chloride, vitamin B 12, an anti-oxidant, a receptor tyrosine kinase growth factor, and a series of growth supporting substances. The specific ingredients are shown in Table 2, in which CHIR99021 is absent.
iCD1 complete culture medium is one of the culture media described in the present invention, and is formed by mixing the supplements as shown in
Furthermore, unless specifically stated otherwise, somatic cells reprogramming based on mice are carried out by employing the following process.
Somatic cell type used in reprogramming is OG2 MEFs of no more than 3 passages. OG2 mice are transgenic mice with green fluorescent protein (GFP) gene linked after the promoter of Oct4 gene specifically expressed by stem cell. At a later stage of the reprogramming, endogenous Oct4 of OG2 MEF is activated, and the green fluorescent protein is concomitantly expressed, so the cells or reprogrammed clones appear green when observed under a fluorescence microscope. The reprogramming efficiencies under different conditions can be compared by a research by directly counting the reprogrammed clones, that is, the green fluorescent clones under a fluorescence microscope, or by analyzing the ratio of the green fluorescent cells through flow cytometry (FCM).
Cell assay and preparation of viruses are as follows. Cells are seeded at a density of 2×104 cells/well in a 12-well attached cell culture plate. 6-18 hrs after the cell seeding, the cells are infected with the viruses carrying a mouse reprogramming factor according to the cell density and state. The infection is carried out in two rounds, 24 hrs after the first round of infection, the second round of infection is carried out, and the virus fluid is replaced by various test media 24 hrs after the second round of infection. The day of replacing the virus fluid is recorded as day 0 (D0), and the GFP fluorescent clones are counted in the original wells or the ratio of the GFP fluorescent cells are analyzed by flow cytometry (FCM) at different times after infection as desired.
The preparation of virus includes transfecting virus packaging cells (PlatE) with reprogramming factor plasmid cloned onto a PMX vector, refreshing the culture media 12-16 hrs after infection, using the culture collected 48 hrs after the transfection as a virus fluid for the first infection, and using the additional culture collected 24 hrs after adding fresh culture as a virus fluid for the second infection.
In addition, unless specifically stated otherwise, some abbreviations should be understood as follows.
SKO specifically refers to SOX2, KIF4, and OCT4 viruses, or to SOX2, KIF4, and OCT4 virus-infected cells, and has the same meaning as three factors. OK specifically refers to OCT4 and KIF4 viruses, or to OCT4 and KIF4 virus-infected cells, and OS specifically refers to OCT4 and SOX2 viruses, or to OCT4 and SOX2 virus-infected cells.
SKO viruses were mixed at 1:1:1 (0.5 ml each), and then 2×104 fibroblasts in total in a well of a 12-well plate were infected, and cultured at 37° C. under 5% CO2 respectively in mES culture medium, mES supplemented with vitamin C, KSR-BN culture medium, and Basal 3.0 culture medium. At day 10 after infection, the reprogrammed clones were directly counted under a fluorescence microscope, and the ratio of the reprogrammed cells was analyzed through flow cytometry.
In order to determine the effects of the ingredients in Basal3.0 on reprogramming under three-factor infection conditions and the optimum working concentration ranges thereof, different concentrations of different ingredients in Basal3.0 are used in iPS efficiency test. Both reprogrammed clone number and reprogrammed cell ratio are used as evaluation indexes, so as to accurately determine the effects of the test substances on the reprogramming.
In concentration test of the ingredients in Basal3.0, the high effect of lithium chloride on reprogramming implicates that inhibition of the glycogen synthase kinase may be a factor promoting reprogramming. Therefore, a series of glycogen synthase kinase inhibitors including CHIR99021, BIO, and SB31xxxx are selected as candidates for the iPS test (two indexes the number of the reprogrammed clones and the ratio of the reprogrammed cells are used as evaluation indexes of reprogramming efficiency). As a result, the GSK3-β inhibitor CHIR99021 can significantly improve the reprogramming efficiency of Basal3.0, and thus the modified Basal3.0, that is, the chemically defined culture medium supplemented with (without limitation) the glycogen synthase kinase-3 (GSK3-β) inhibitor CHIR99021 is named as iCD1.
Through the iPS efficiency test, reprogramming efficiencies of iCD1 and all advanced culture schemes currently reported are compared, in which the advanced culture schemes include mES supplemented with vitamin C, mES to KSR (that is, at day 4 after infection, a classical embryonic stem cell culture medium (mES) is converted to a commercialized serum-free culture medium (KSR)), and KSR-BN.
In order to more objectively compare the efficiencies under the several different culture conditions, the tests of the induced reprogramming efficiencies of KSR-BN, mES supplemented with vitamin C, and mES to KSR were repeated according to the report in literatures or patents, and the reprogramming efficiencies at different days are calculated.
In order to evaluate the effects of main ingredients in iCD1 on the reprogramming efficiency, the iPS efficiency tests were carried out with iCD1 without vitamin C, bFGF, CHIR99021, lithium chloride, vitamin B12, and thiamine respectively or entirely, in which the iCD1 culture medium was used as a complete control, and the mES culture medium was used as a basic reference. The number of the reprogrammed clones at day 8 after infection are as shown in
In order to determine whether the effects of the above six substances on reprogramming are cell growth dependent or independent, growth curves of the embryonic fibroblasts infected with reprogramming factor SKO and then cultured respectively in 9 culture states including iCD1, iCD1 without vitamin C, iCD1 without bFGF, iCD1 without CHIR99021, iCD1 without lithium chloride, iCD1 without vitamin B12, iCD1 without thiamine, iCD1 without all of the six ingredients, and mES were plotted. 2×104 initial Oct4-GFP MEFs were infected with SKO viruses, and then cultured respectively in the above 9 culture media. The cells were digested respectively at the day of infection and medium replacement (day D0) and days 3 and 6 after infection, and the cells per well were counted. The replications n in each group of test are 3, and the error bar represents standard deviation (SD). The result shown in
In order to further determine the optimum concentration of bFGF affecting reprogramming, different doses of bFGF were added to iCD1, to detect the effect on reprogramming.
In order to testify that other receptor tyrosine kinase family growth factors also play a role in reprogramming, epidermal growth factor (EGF) was also used in the reprogramming efficiency test. Oct4-GFP MEFs were infected with SKO viruses, and then cultured respectively in iCD1 culture media without receptor tyrosine kinase, supplemented with bFGF, and EGF.
In order to further improve the iCD1 reprogramming efficiency, previously reported several substances able to improve the reprogramming efficiency, such as an ALK5 inhibitor A83-01, a histone methylase inhibitor valproic acid (VPA), and fetal bovine serum conventionally used for mouse embryonic stem cells, were added to iCD1 for the reprogramming efficiency test.
In order to discuss the effect of the initial cell density on the reprogramming efficiency, Oct4-GFP transgenic MEFs were subjected to two rounds of infection with SKO viruses, digested with trypsin, and seeded in a 96-well plate at different cell densities including specifically 5, 10, 50, 100, 200, 500, 1000, 2000, 5000, and 1×104 cells/cm2. After 8 days of culture in iCD1, the reprogramming efficiencies at various different seeding densities were calculated as a ratio the number of the reprogrammed clones at day 8 to the initial number of the seeded cells. The replications n in the test are 6, and the error bar represents standard deviation (SD).
In order to verify that the iCD1 culture system is superior to other culture system at a molecular level, relative expression levels of the pluripotent molecular marker Nanog and endogenous Oct4 specifically expressed by stem cells were used to evaluate the progress of the whole cell reprogramming process. Specific implementation method was as follows. Oct4-GFP transgenic MEFs were subjected to two rounds of infection with SKO viruses, and then cultured respectively in mES culture medium, KSR-BN culture medium, and iCD1 culture system. The day of infection and medium replacement was recorded as day 0, the culture samples were collected respectively after 2, 4, 6, and 8 days of culture in the mES culture medium, KSR-BN culture medium, and iCD1 culture medium, the ribonucleotides were extracted from the samples, and the expression levels of nanog and endogenous Oct4 were detected by real-time fluorescent quantitative polymerase chain reaction (RT-PCR). The test result is as shown in
b shows the expression results of the pluripotent molecular markers verified at a protein level. Oct4-GFP MEFs were infected with SKO viruses, cultured in iCD1 for 8 consecutive days, and directly fixed on a cell culture plate for immunofluorescence assay. The result shows that at day 8, the pluripotent molecular markers Cdh1, SSEA-1, and Nanog are all expressed in the reprogrammed clones appeared in the plate. The scale shown is 100 microns.
Silence of the exogenous transcription factor is an important event at the later stage of reprogramming, and also an index for determining complete reprogramming. In order to more intuitively tracking the silence of the exogenous transcription factor, red fluorescent protein Ds-Red was used to simulate the silence of the exogenous transcription factor. Specific implementation method was as follows. Oct4-GFP transgenic MEFs were infected with red fluorescent protein Ds-Red and SKO viruses, cultured in iCD1 culture medium, and tracked under a fluorescence microscope to observe the expression of green fluorescence (indicating the expression of the endogenous pluripotent molecular marker Oct4) and red fluorescence (indicating the expression of exogenous viruses).
In order to further verify that the green fluorescence positive and red fluorescence negative cell population is more highly programmed than red and green fluorescence positive cell population and green fluorescence negative and red fluorescence positive cell population, the three cell populations in 8-day culture under the iCD1 culture condition were sorted out by a sorting flow cytometer.
Capability of forming chimeric mice is a very critical standard characteristic of mouse embryonic stem cells. In order to verify in a more strict sense that the reprogrammed clones have no difference from the embryonic stem cells, the reprogrammed cells picked through different methods were used in construction of chimeric mice. Specific method was as shown in
Under iCD1 culture condition, the extremely high reprogramming efficiency with the three SKO transcription factors indicates that reprogramming into somatic cells may be achieved with fewer transcription factors. Based on this assumption, different combinations of viruses (ok/os/sk) were used to infect mouse fibroblasts, and a specific method was as follows. OCT4 and KIF4 viruses were mixed at 1:1 (0.5 ml each), and infected 2×104 cells/well in a 12-well plate; OCT4 and SOX2 viruses were mixed at 1:1 (0.5 ml each), and infected 3×104 cells/well in a 12-well plate; and KIF and Sox2 viruses were mixed at 1:1 (0.5 ml each), and infected 3×104 cells/well in a 12-well plate. The three groups of infected cells were cultured respectively in the iCD1 culture medium or the conventional mES culture medium, continuously cultured and observed.
The fact that the reprogrammed clones can be generated in the iCD1 with OK and OS suggests that Oct4 plays a key role in the reprogramming process, and thus attempts are made to investigate whether the reprogrammed clones can be generated in the iCD1 with Oct4 alone. 3×104 Oct4-GFP transgenic MEFs were subjected to two rounds of infection with Oct4 viruses, and cultured in iCD1 for about 30 consecutive days. At this time, there was still no fluorescent clones appeared. The culture was digested with trypsin, subcultured on feeder cells, and continuously cultured in iCD1. At about day 5 after passage, green fluorescent reprogrammed clones appeared, as shown in
Oct4-vp16 is Oct4 gene after which vp16 sequence is linked, co-expression thereof can enhance the transcription capability of Oct4. It is confirmed through experiments that the capability of Oct4-vp16, Sox2, and Kif4 for reprogramming somatic cells is higher than that of Oct4, Sox2, and Kif4. In order to verify that iCD1 is superior to other culture conditions in the reprogramming process in which Oct4-vp16 is involved, 2×104 Oct4-GFP MEFs were infected with Oct4-vp16, Sox2, and Kif4, and then cultured respectively in mES, mES supplemented with vitamin C, and iCD1 culture system. At day 10 after infection, the reprogrammed clones were calculated. The replications n in each group of test are 3, and the error bar represents standard deviation (SD). The result is shown in
Reprogramming into adult stem cells can be achieved with Oct4 under iCD1 condition; however the efficiency is very low, and subculture is required. Whether Oct4-vp16 can improve the reprogramming efficiency of a single factor is highly concerned due to the properties of Oct4-vp16. In order to detect this, 3×104 Oct4-GFP transgenic MEFs were infected with Oct4-vp16 viruses, and then cultured respectively in mES, mES supplemented with vitamin C, and iCD1 culture medium. As a result, it was found that at about day 10 after infection, green fluorescent reprogrammed clones appeared in the wells containing the iCD1 culture medium, as shown in
The above tests strongly demonstrate that in reprogramming by using Oct4-vp16, the iCD1 culture medium is obviously superior to other culture media.
In order to test the use of the iCD1 core ingredients in serum, iCD1 core ingredients, vitamin C, lithium chloride, bFGF, insulin, and glycogen synthase kinase 3-b inhibitor CHIR99021 were added to mES, and a culture medium thus prepared is referred to as SmES (Super-mES). The reprogramming effects achieved with SKO and common Oct4 in SmES were detected. mES culture medium and mES culture medium supplemented with vitamin C were used as control. The result is as shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 201010167062.3 | Apr 2010 | CN | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CN10/75551 | 7/29/2010 | WO | 00 | 10/11/2011 |
