CN115772505A - Culture medium and method for promoting reprogramming of somatic cells into induced pluripotent stem cells - Google Patents
Culture medium and method for promoting reprogramming of somatic cells into induced pluripotent stem cells Download PDFInfo
- Publication number
- CN115772505A CN115772505A CN202310101650.4A CN202310101650A CN115772505A CN 115772505 A CN115772505 A CN 115772505A CN 202310101650 A CN202310101650 A CN 202310101650A CN 115772505 A CN115772505 A CN 115772505A
- Authority
- CN
- China
- Prior art keywords
- cells
- culture medium
- reprogramming
- pluripotent stem
- induced pluripotent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention provides a culture medium and a method for promoting reprogramming of somatic cells into induced pluripotent stem cells, wherein the method comprises the steps of introducing a plasmid containing a reprogramming factor into the somatic cells through an electrotransfer introduction mode, and then adding small chemical molecules TTNPB, 2-PCPA, repsox and UNC0379 in stages in the culture medium to promote reprogramming of the somatic cells. The method can improve the reprogramming efficiency of the urine cells by about 7 times, shorten the reprogramming time by 4-5 days, and improve the reprogramming efficiency of the blood cells by about 2.5 times. In addition, the culture medium is compatible with the characteristics of urine cells and peripheral blood cells, so that the culture medium has wider applicability.
Description
Technical Field
The invention relates to the technical field of induced pluripotent stem cells, in particular to a culture medium and a method for promoting reprogramming of somatic cells into induced pluripotent stem cells.
Background
With the rapid development of stem cell biology in recent 20 years, various types of somatic cells can realize cell fate remodeling through a reprogramming technology, so that induced pluripotent stem cells (iPS) with unlimited proliferation capacity and multidirectional differentiation potential are formed. Among different somatic cell types, urine cells and peripheral blood cells have the characteristics of easiness in obtaining, no age limit and low ethical constraint, are particularly suitable for serving as reprogrammed seed cells and serve the wide demand of future precise medical treatment.
According to literature reports, the method of inducing a urinary cell reprogramming system is derived from a reprogramming scheme established in the Paniculatus pellitis problem group and the Miguel A. Esteban problem group (Zhou T, benda C, duzinger S, et al. Generation of induced plosive stem cells from urea [ J ]. Journal of the American Society of neurology, 2011, 22 (7): 1221-1228.); however, this scheme has short plates with low reprogramming efficiency, long preparation period, and the like. In order to solve these problems, in recent years, the reprogramming system based on urine cells has been optimized by people such as zhuhuang libei et al, for example, by introducing a new transcription factor capable of promoting reprogramming efficiency into the original nuclear transcription factor system, or by adding a small molecule inhibitor capable of promoting the early stage of reprogramming into the reprogramming culture medium system, so that the reprogramming efficiency of urine cells is improved from 0.001% to about 0.01%, but the system still has a large optimization space, and in particular, the reprogramming efficiency and the induction time are still to be improved. In 2010, cell Stem Cell simultaneously reports the work of three research groups to reprogram human peripheral blood cells to obtain iPSCs, and in recent years, kunisato, shujunying and the like are optimized from different aspects in a peripheral blood Cell reprogramming system, so that the reprogramming efficiency is improved to about 2.5% from the initial 0.0008%. However, no medium has been reported which is compatible with reprogramming of peripheral blood mononuclear cells and urine cells.
Disclosure of Invention
In view of the above, the present invention provides a culture medium and a method for promoting reprogramming of somatic cells into induced pluripotent stem cells, which can be applied to different transcription factor inducing systems and somatic cell types, such as urine cells, peripheral blood cells, etc.; the method can improve the reprogramming efficiency of urine cells by about 7 times, shorten the induction time by 4-5 days, and improve the reprogramming efficiency of peripheral blood by about 2.5 times.
In order to achieve the purpose, the technical scheme of the invention is realized as follows:
a method of promoting reprogramming of somatic cells to induced pluripotent stem cells, the method comprising the steps of:
1) Introducing a plasmid containing a reprogramming factor into a somatic cell by an electrotransfer method;
2) Culturing in culture medium containing additives TTNPB, 2-PCPA and Repsox for 7-8 days;
3) The culture is continued for 6-7 days in a culture medium containing additives TTNPB, 2-PCPA and UNC0379.
Further, TTNPB concentration is 0.5-5. Mu.M, 2-PCPA concentration is 10-20. Mu.M, repsox concentration is 0.5-5. Mu.M, and UNC0379 concentration is 1-5. Mu.M.
Further, the medium of step 2) comprises a base medium consisting of 1% GlutaMax, 1% NEAA, DMEM-F12%.
Further, the culture medium of the step 3) comprises a basic culture medium and additives, wherein the basic culture medium is mTeSR1, and the additives further comprise 1-5 mu M CHIR99021, 0.5-3 mu M PFT-alpha, 10-15 mu M Y-27632, 100-500 mu M NaB and 0.1-1 mu M PD0325901.
Further, reprogramming factors include OCT4, SOX2, KLF4, and C-Myc.
Further, the plasmids were 2 to 5. Mu.g of PCEP4-OCT4-IRES-SOX2 plasmid, 0.5 to 2. Mu.g of PCEP4-KLF4 plasmid and 0.5 to 2. Mu.g of PCEP4-C-Myc plasmid.
The invention also provides a culture medium used for the method, wherein the culture medium comprises a culture medium A and a culture medium B, the culture medium A comprises additives TTNPB, 2-PCPA and Repsox, and the culture medium B comprises additives TTNPB, 2-PCPA and UNC0379.
The invention also provides an induced pluripotent stem cell constructed by the method.
The invention also provides application of the induced pluripotent stem cells in establishing a disease model.
Compared with the prior art, the culture medium and the method for promoting the reprogramming of the somatic cells into the induced pluripotent stem cells have the following advantages:
the culture medium for promoting the reprogramming of the somatic cells into the induced pluripotent stem cells can be suitable for different transcription factor induction systems and somatic cell types, such as urine cells, peripheral blood cells and the like; compared with the existing scheme, the method has the advantages that the efficiency is higher, the reprogramming efficiency of the urinary cells is improved by about 7 times, and the reprogramming efficiency of the peripheral blood cells is improved by about 2.5 times; the reprogramming induction cycle of the urine cells takes shorter time, and is shortened from the original 16-21 days to 13-15 days.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate an embodiment of the invention and, together with the description, serve to explain the invention and not to limit the invention. In the drawings:
FIG. 1 is a graph of the morphological changes of urine cells during reprogramming; (a) is a urine cell morphology map; (b) a urine cell morphology map for Day 0; (c) is a cell morphology map of Day 9; (d) is a cell morphology map of Day 14; (e) obtaining a morphology map of the induced pluripotent stem cells;
FIG. 2 is a graph of the morphological changes of peripheral blood cells during reprogramming; (a) is a peripheral blood cell morphology map; (b) is a peripheral blood cell morphology map of Day 0; (c) is a cell morphology map of Day 6; (d) is a cell morphology map of Day 14; (e) obtaining a morphology map of the induced pluripotent stem cells;
FIG. 3 is a graph of immunofluorescence results on induced pluripotent stem cells reprogrammed by urinary cells; (a) is a NANOG + SOX2+ DAPI staining result stack, (b) is a staining result of the marker NANOG, (c) is a staining result of the marker SOX2, (d) is a DAPI staining result, (e) is a SSEA4+ OCT4+ DAPI staining result stack, (f) is a staining result of the marker SSEA4, (g) is a staining result of the marker OCT4, and (h) is a DAPI staining result;
FIG. 4 shows the results of flow cytometry assays for the induced pluripotent stem cell surface antigens TRA-1-60 and TRA-1-81;
FIG. 5 is the results of an in vitro three germ layer differentiation assay of induced pluripotent stem cells; (a) is a morphological diagram of induction and differentiation of iPSC to myocardial cells, (b) is an mRNA expression level of myocardial cell markers NKX2.5, TNNT2 and ACTN2, (c) is a morphological diagram of induction and differentiation of iPSC to neuronal cells, (d) is an mRNA expression level of neuronal cell markers SOX1, PAX6 and Nestin, (e) is a morphological diagram of induction and differentiation of iPSC to liver cells, and (f) is an mRNA expression level of liver cell markers AFP and ALB;
FIG. 6 is the results of an in vivo three germ layer differentiation assay of induced pluripotent stem cells; (a) teratomas obtained after implanting ipscs subcutaneously in mice, (b) - (g) H & E staining results of teratoma endoderm sections, (b) and (c) original glandular tubes representing endoderm, (d) muscle tissue representing mesoderm, (E) adipose tissue representing mesoderm, (f) pigment epithelial tissue representing ectoderm, (g) neurorosette tissue representing ectoderm;
FIG. 7 shows the karyotype detection results for the P20 generation of iPS;
FIG. 8 is the final alkaline phosphatase staining results after addition of different small molecules at S1 or S2, presented as two independent replicates, with more than three actual replicates;
FIG. 9 is a statistical graph of the number of cell clones cultured in S1 stage or S2 stage with different small molecules added;
FIG. 10 is a statistical plot of the number of cell clones in the S1 stage and S2 stage under different small molecule combination schemes (a-e) after four small molecules have been selected;
FIG. 11 is a graph showing the results of alkaline phosphatase staining at the S1 stage and S2 stage under different small molecule combination schemes (a-e) after four small molecules have been selected;
FIG. 12 shows the result of alkaline phosphatase staining of urine cells in 4 groups in the culture medium of the control group and the experimental group;
FIG. 13 is a cell morphology chart of control group and experimental group numbered 1 in the urine cell reprogramming system at Day8 and Day 14;
FIG. 14 shows the results of alkaline phosphatase staining of peripheral blood cells in the culture medium of the control group and the experimental group;
fig. 15 is a statistical chart of iPSC clone numbers of urine cells and peripheral blood cells in the culture medium of the control group and the experimental group.
Detailed Description
The invention will be described in detail below with reference to the drawings and examples.
The invention provides a method for promoting reprogramming of somatic cells into induced pluripotent stem cells, which comprises the following steps:
1) Introducing a plasmid containing a reprogramming factor into a somatic cell by an electrotransfer method;
2) Culturing in culture medium containing additives TTNPB, 2-PCPA and Repsox for 7-8 days, specifically for 7 days;
3) Culturing in culture medium containing additives TTNPB, 2-PCPA and UNC0379 for 6-7 days, specifically for 7 days.
Wherein, the concentration of TTNPB is 0.5-5 μ M, the concentration of 2-PCPA is 10-20 μ M, the concentration of RepSox is 0.5-5 μ M, and the concentration of UNC0379 is 1-5 μ M.
Preferably, TTNPB is at a concentration of 1. Mu.M, 2-PCPA is at a concentration of 15. Mu.M, repsox is at a concentration of 5. Mu.M, and UNC0379 is at a concentration of 1. Mu.M.
Specifically, the medium of step 2) comprises a base medium of DMEM-F12 containing 1% GlutaMax, 1% NEAA, 10% FBS and additives further comprising 1-5. Mu.M CHIR99021, 0.5-3. Mu.M PFT-alpha, 5-15. Mu.M Y-27632, 100-500. Mu.M NaB; preferably, the additives include 3. Mu.M CHIR99021, 1. Mu.M PFT-alpha, 10. Mu.M Y-27632, 250. Mu.M NaB;
the culture medium of the step 3) comprises a basic culture medium and additives, wherein the basic culture medium is mTeSR1, and the additives further comprise 1-5 mu M CHIR99021, 0.5-3 mu M PFT-alpha, 10-15 mu M Y-27632, 100-500 mu M NaB and 0.1-1 mu M PD0325901; preferably, the additives include 3. Mu.M CHIR99021, 1. Mu.M PFT-. Alpha., 10. Mu.M Y-27632, 250. Mu.M NaB, 0.5. Mu.M PD0325901.
Reprogramming factors include OCT4, SOX2, KLF4, and C-Myc.
Specifically, the plasmids are 2-5 μ g of PCEP4-OCT4-IRES-SOX2 plasmid, 0.5-2 μ g of PCEP4-KLF4 plasmid and 0.5-2 μ g of PCEP4-C-Myc plasmid; preferably, 4. Mu.g of the PCEP4-OCT4-IRES-SOX2 plasmid, 2. Mu.g of the PCEP4-KLF4 plasmid and 2. Mu.g of the PCEP4-C-Myc plasmid are used as the plasmids.
Example 1 reprogramming of urine cells to induce pluripotent stem cells
1) Obtaining somatic cells to prepare for electrotransformation: collecting urine cells, amplifying, digesting urine cells with 0.25% pancreatin, counting, and collecting 1.2-1.5 × 10 6 4 mu g of PCEP4-OCT4-IRES-SOX2 plasmid, 2 mu g of PCEP4-KLF4 plasmid and 2 mu g of PCEP4-C-Myc plasmid are simultaneously introduced into each urine source Cell by an electrotransfer mode, and the cells are planted in a culture plate coated with matrigel 30min in advance after the electrotransfer is completed, wherein the culture Medium is a urinary Cell maintenance Medium (Renal Epithelial Cell Growth Medium).
2) After 24h, change to S1 stage medium A (Day 1-7):
the culture medium comprises a basal medium and additives, wherein the basal medium is DMEM-F12 containing 1% by volume of GlutaMax, 1% by volume of NEAA and 10% by volume of FBS, and the additives comprise 3 mu M of CHIR99021, 1 mu M of PFT-alpha, 10 mu M of Y-27632, 250 mu M of NaB, 1 mu M of TTNPB, 15 mu M of 2-PCPA and 5 mu M of Repsox; during which the medium was changed daily.
3) When a plurality of compact circular clone islands are present in the plate, the plate is changed to S2 stage medium B (Day 8-14):
the culture medium comprises a basic culture medium and additives, wherein the basic culture medium is mTeSR1, and the additives comprise 3 mu M CHIR99021, 1 mu M PFT-alpha, 10 mu M Y-27632, 250 mu M NaB, 0.5 mu M PD0325901, 15 mu M2-PCPA, 5 mu M Repsox and 1 mu M UNC0379; during which the medium was changed daily.
By day14 of induced differentiation, the urine cell-derived reprogramming group can obtain an inducible stem cell clone.
Example 2 reprogramming of peripheral blood cells to induce pluripotent stem cells
1) Obtaining somatic cells to prepare for electroporation: collecting peripheral blood cells, amplifying, digesting peripheral blood cells with 0.25% pancreatin, counting, and collecting 2-5 × 10 6 4. Mu.g of PCEP4-OCT4-IRES-SOX2 plasmid, 2. Mu.g of PCEP4-KLF4 plasmid, and 2. Mu.g of PCE were simultaneously introduced into each peripheral blood cell by electroporationThe P4-C-Myc plasmid, after electrotransfer was completed, was plated on a plate coated with matrigel 30min in advance, and the culture Medium was peripheral blood Cell maintenance Medium containing 100ng/ml SCF, 10ng/ml IL-3, 2U/ml EPO, 20ng/ml IGF-1, 1. Mu.M dexamessone, 0.2mM 1-thioglycolol Hematotic Stem Cell Expansion Medium.
2) After 24h, change to S1 stage medium A (Day 1-7):
the culture medium comprises a basal medium and additives, wherein the basal medium is DMEM-F12 containing 1% by volume of GlutaMax, 1% by volume of NEAA and 10% by volume of FBS, and the additives comprise 3 mu M of CHIR99021, 1 mu M of PFT-alpha, 10 mu M of Y-27632, 250 mu M of NaB, 1 mu M of TTNPB, 15 mu M of 2-PCPA and 5 mu M of Repsox; during which the medium was changed daily.
3) When multiple compact circular clonal islands are present in the plate, medium B (Day 8-14) is changed to S2 stage:
the culture medium comprises a basic culture medium and additives, wherein the basic culture medium is mTeSR1, and the additives comprise 3 mu M CHIR99021, 1 mu M PFT-alpha, 10 mu M Y-27632, 250 mu M NaB, 0.5 mu M PD0325901, 15 mu M2-PCPA, 5 mu M Repsox and 1 mu M UNC0379; during which the medium was changed daily.
By day14 of induced differentiation, the peripheral blood cell-derived reprogramming group can obtain an inducible stem cell clone.
Example 3 pluripotency assay
Taking the example of the induced stem cells derived from urine cells in example 1, the stem cell performance was determined.
1) Alkaline phosphatase staining
And (3) fixing the sample by 4% paraformaldehyde, washing by PBS, and dyeing according to the operation flow of the kit.
2) Immunofluorescence assay
After being fixed by 4% paraformaldehyde, permeabilized by 0.5% TritonX100 and sealed by 5% donkey serum, the cell nucleus is marked by a first antibody and a second antibody of a pluripotency marker respectively, and is marked by DAPI (Dairy amplified Polyacrylamide), imaged and analyzed under a fluorescence microscope.
3) Flow cytometry characterization
Using Accutase to digest cells, counting the cells, and taking 1-3 × 10 6 Individual single cells; after it is fixed and sealed, it is labeled with a fluorescent antibody as a pluripotent marker, and the positive ratio is analyzed by a flow cytometer.
4) In vitro three germ layer differentiation assay (iPS directed differentiation test)
i) Mesoderm differentiation method (cardiomyocyte induction):
induced differentiation of iPS into cardiomyocytes was achieved using the following differentiation media in order:
day1: adding 5 μ M CHIR99021 into RPMI1640 containing 2% B27, and culturing;
day2: adding 0.6U/ml heparin into RPMI1640 containing 2% B27, and culturing;
day3-5: adding 3 μ M IWP2 and 0.6U/ml heparin to RPMI1640 containing 2% B27, and culturing;
day6-7: adding 0.6U/ml heparin into RPMI1640 containing 2% B27, and culturing;
day8-14, cultured in RPMI1640 containing 2% B27; during which the medium was changed daily.
ii) ectodermal differentiation method (neuronal cell induction):
the following differentiation media were used in sequence to achieve induction differentiation of iPS into neuronal cells:
day1-7: adding 10ng/ml LIF, 3. Mu.M CHIR99021, 2. Mu.M SB431542, 0.1. Mu.M Compound E to a medium containing 5. Mu.g/ml BSA, 2% B27, 1% N2, 47% DMEM-F12 and 50% NBM for culturing; during which the medium was changed daily.
iii) Endoderm differentiation method (liver cell induction):
induced differentiation of iPS into hepatocytes was achieved using the following differentiation media in order:
day1: RPMI1640 containing 2% B27 was cultured in the presence of 100ng/ml of ActA and 3. Mu.M of CHIR 99021;
day2-3: RPMI1640 containing 2% B27 was cultured in the presence of 100ng/ml of ActA;
day4-8: culturing the cells in KO-DMEM containing 20% of KSR with 1% DMSO and 0.1 mM. Beta. -ME;
day9-21: adding 20ng/ml HGF, 10ng/ml OSM, 0.5. Mu.M DEX and 10. Mu.M HH to HCM, and culturing; during which the medium was changed daily.
5) In vivo three germ layer differentiation assay (teratoma nodulation test)
Taking the cells obtained by reprogramming, digesting with Accutase, taking 1 × 10 6 After the individual cells were resuspended in Matrigel-DMEM mixture (1 mix), they were inoculated subcutaneously into immunodeficient mice (NOD-SCID mice, female, SPF grade).
After 2 months, the plants were dissected in situ, fixed, dehydrated, and paraffin embedded, wax blocks were sectioned (3.5 μ M) using a microtome, and the differentiation of the three germ layers was examined separately based on the histiocyte characteristics of the plants using H & E staining techniques.
6) Karyotype detection
After 0.4% colchicine is used for incubating iPS cells in logarithmic growth phase for 4h, potassium chloride solution is used for processing, paraformaldehyde is used for continuous fixation, and the proportion of dividing phase cells is 10% -20% preferably.
Comparative example 1 comparison of different Small molecule additives
Among the many known small molecule compound libraries, the present invention selects 9 small molecule compounds, as shown in table 1, and considers that each small molecule compound has different time to act in the reprogramming process, except for the optimal concentration, the time window plays an important role, and 9 different small molecules in table 1 are added respectively, the initial cells in the screening test are selected from human urine source cells, the S1 stage is set for 10 days, the S2 stage is set for 8 days, the 18 th day is stained with alkaline phosphatase, and the number of positive clones is compared.
The S1 stage basal medium comprises: adding 3 μ M CHIR99021, 1 μ M PFT- α, 10 μ M Y-27632, 250 μ M NaB and 0.5 μ M A83-01 into DMEM-F12 containing 10% FBS;
the S2 stage basal medium comprises: mu.M CHIR99021, 1. Mu.M PFT-. Alpha.10. Mu.M Y-27632, 250. Mu.M NaB, 0.5. Mu.M A83-01, 0.5. Mu.M PD032590 were added to mTeSR 1.
TABLE 1 list of small molecules
| Name of Small molecule | Function of | Dosage of | |
| SB431542 | TGF-beta |
5 μM | |
| VPA | Histone deacetylase inhibitors | 250 μM | |
| TTNPB | RAR signaling |
1 μM | |
| 3-DZNEP | Histone methyltransferase inhibitors | 0.5 μM | |
| UNC0379 | |
1 μM | |
| 5-Aza | |
5 μM | |
| Linifanib | VEGFR/ |
1 μM | |
| 2- | LSD1 inhibitors | 15 μM | |
| RepSox | TGF-beta |
5 μM |
Comparative example 2 reprogramming comparison of urine cells and peripheral blood cells
On the basis of determining the small molecule additive, urine cells and peripheral blood cells are respectively cultured in a control way.
1) Urine cells:
urine cell reprogramming control group medium:
stage S1 is (Day 0-Day 10): adding 3 μ M CHIR99021, 1 μ M PFT- α, 10 μ M Y-27632, 250 μ M NaB and 0.5 μ M A83-01 into DMEM-F12 containing 10% FBS;
stage S2 is (Day 11-Day 17): mu.M CHIR99021, 1. Mu.M PFT-. Alpha.10. Mu.M Y-27632, 250. Mu.M NaB, 0.5. Mu.M A83-01, 0.5. Mu.M PD032590 were added to mTeSR 1.
2) Peripheral blood cells:
peripheral blood cell reprogramming control group medium:
s1 stage (Day 0-Day 6) adding 1% GlutaMax, 1% NEAA, 50ng/ml FGF2, 1% ITS, 50 μ g/ml VC in Knockout DMEM/F12;
s2 stage (Day 7-Day 14) 250. Mu.M Sodium butyrate was added to mTeSR 1.
Results and analysis:
1. FIGS. 1 and 2 are graphs showing the morphological changes of urine cells and peripheral blood cells during reprogramming, respectively, and it can be seen that induced pluripotent stem cells were obtained over 14 days. Moreover, the immunofluorescence results in fig. 3 prove that most of the cells obtained by the reprogramming method highly express the pluripotency markers OCT4, SOX2, SSEA4 and NANOG; figure 4 flow cytometry results show that: the positive rates of the stem cell surface antigens TRA-1-60 and TRA-1-81 are 99.1 percent and 98.4 percent respectively. These results suggest the potential for these cells to differentiate divergently.
Meanwhile, the culture medium provided by the invention can be used for urine cells and peripheral blood cells.
2. To confirm the multipotentiality, the differentiation was examined by in vitro three germ layer differentiation (directional differentiation assay) and in vivo three germ layer differentiation (teratoma assay), respectively:
1) In the in vitro three-germ layer differentiation assay, cardiomyocytes, neuronal cells, and hepatocytes, which are representative cell types of mesoderm, ectoderm, and endoderm, respectively, were induced to differentiate; along with the induction, the fluorescent quantitative PCR result shows that: the cardiomyocyte-specific markers TNNT2 and ACTN2 (fig. 5 (b)), the neuronal cell-specific marker PAX6 (fig. 5 (d)), and the hepatocyte marker ALB (fig. 5 (f)) were continuously elevated; finally, typical morphological characteristics of three cells are presented, as shown in (a) of fig. 5, short-stub-like, self-expanding cardiomyocytes; as shown in fig. 5 (c), neuronal cells having inclusion and synapses; as shown in FIG. 5 (e), the liver cells were pebbly-shaped and partially double-nucleus. These results demonstrate the ability of the three germ layers to differentiate in vitro.
2) In the in vivo three germ layer differentiation assay, the cells formed tumor-like tissues 2 months after subcutaneous implantation in mice, and had a length of 8.53mm and a width of 6.25mm, as shown in FIG. 6 (a).
The H & E staining results of (b) - (g) panels in FIG. 6 show that: the tissue is a complete teratoma with endodermal (primitive ductal epithelial cells), mesodermal (muscle cells, adipocytes) and ectodermal (nerve cells) structures, and these results demonstrate the ability of the three germ layers to differentiate in vivo.
3) As shown in figure 7, the results of the karyotype assay show: the iPS continuously amplified for 20 generations still maintains normal karyotype. The stability of continuous passage amplification of the iPS is demonstrated, and no mutation phenomenon occurs.
3. FIG. 8 shows the final alkaline phosphatase staining results after 9 small molecules were applied to the S1 and S2 stages, respectively, showing two independent replicates, with more than three actual replicates; FIG. 9 is a statistical plot of the number of clones from the results of multiple independent experimental screens.
As can be seen from the combination of FIG. 8 and FIG. 9, the compounds TTNPB, 2-PCPA and Repsox have the function of promoting reprogramming when added in the S1 stage, wherein the 2-PCPA promoting effect is most obvious; the compounds RepSox, 2-PCPA and UNC0379 have promoting effects when added at the S2 stage, and the promoting effects of the same 2-PCPA are most obvious at the S2 stage.
To verify the effect of these four small molecule compounds (Repsox, UNC0379, 2-PCPA, TTNPB) at different stages S1 and S2, we screened these four compounds in different combinations, while listing five different schemes (a-e) according to the results of TTNPB addition at stage S1 and UNC0379 at stage S2, as shown in Table 2, the results are shown in FIG. 10 and FIG. 11. The final result shows that the clone quantity is the largest when TTNPB, 2-PCPA and Repsox are added together in the S1 stage, and when the three small molecules are added simultaneously, the three small molecules have no toxicity to cells and cannot generate inhibition effect, and the combined promoting effect is better than the effect of single addition; when UNC0379, 2-PCPA and Repsox are added together at the S2 stage, the number of clones is the largest, and similarly, when the three small molecules are added simultaneously, the three small molecules have no toxicity to cells and cannot generate inhibition effect, and the combined promotion effect is better than that of the single small molecules.
Table 2 five different small molecule combination schemes
| Scheme(s) | a | b | c | d | e |
| TTNPB | + | + | + | - | + |
| 2-PCPA | - | + | + | + | + |
| RepSox | - | - | + | + | + |
| UNC0379 | - | - | - | + | + |
4. The S1-stage and S2-stage media obtained after screening were used as the experimental group (i.e., the media used in examples 1 and 2) and the control group (i.e., the control medium given in comparative example 2) for comparison.
The urine cells were cultured in the medium of example 1 as an experimental group, and the control group was the medium of the control group for reprogramming the urine cells in comparative example 2, while the comparison was made in 4 parallel experiments, as the comparison results of numbers 1 to 4 given in fig. 12, and fig. 13 is a cell morphology chart of the number 1 group at Day8, day14, day 18.
As a result, it was found that, in the urine cell reprogramming system, as shown in FIGS. 12 to 13, the number of alkaline phosphatase staining-positive clones was significantly increased, and the number of clones was converted into the reprogramming efficiency, which was the number of clones divided by the number of starting cells, which was 3X 10 4 Per well, 0.008% for the control and 0.065% for the experimental.
In addition, the reprogramming time is also shortened, as shown in FIG. 13, circular cell swelling occurs in the experimental group day8, but not in the control group, and the induced pluripotent stem cell clone is mature in day14, which takes 18 days, 4 days shorter than the control group.
As shown in FIG. 14, the result of alkaline phosphatase staining of peripheral blood cells in the culture medium of the control group (i.e., comparative example 2) and the experimental group (i.e., example 2) was also shown, the efficiency of peripheral blood cells in the culture medium system of the experimental group was improved, and the starting cell number of blood cells was 1.5X 10 5 Per well, the reprogramming efficiency is improved from 0.021% to 0.052%.
FIG. 15 is a statistical graph of the numbers of iPSC clones in the control group and the experimental group of urinary cells and peripheral blood cells, and it can be seen that the reprogramming efficiency is significantly improved.
In conclusion, the method can improve the reprogramming efficiency of the urine cells by about 7 times, shorten the reprogramming time by 4-5 days, and improve the reprogramming efficiency of the blood cells by about 2.5 times. Moreover, the culture medium used by the invention is suitable for urine cells, peripheral blood cells, different transcription factor induction systems and somatic cell types, and the applicability is wide.
TABLE 3 list of reagents
| Name of reagent consumable | Company (goods number) |
| mTeSR1 | STEMCELL (100-0276) |
| RPMI 1640 | Sigma (R8758) |
| DMEM-F12 | Sigma (SCM162) |
| Renal Epithelial Cell Growth Medium (Kidney Epithelial Cell Medium) | LONZA（CC-3190） |
| Hematopoetic Stem Cell Expansion Medium (Hematopoietic Stem Cell Expansion Medium) | Sigma(S0192) |
| KO-DMEM | Gibco (10829018) |
| HCM | LONZA (CC-3199) |
| KSR | Gibco (10828028) |
| FBS | Gibco (16010-159) |
| B27 Supplement | Gibco (17504044) |
| B27 (-insulin) Supplement | Gibco (A1895601) |
| B27 (-Va) Supplement | Gibco (12587010) |
| N2 Supplement | Gibco (17502048） |
| GlutaMax | Gibco (35050061) |
| NEAA | Gibco (11140050) |
| Accutase | Gibco (A1110501) |
| DMSO | Sigma (D2650) |
| β-ME | Sigma (M3148) |
| Act A | R&D (338-AC) |
| LIF | R&D (7734-LF) |
| HGF | R&D (294-HG) |
| OSM | R&D (295-OM) |
| CHIR99021 | Tocris (TB4423) |
| NaB | Tocris (3850) |
| PD0325901 | Tocris (4192) |
| Y-27632 | Tocris (1254) |
| A83-01 | Tocris (2939) |
| PFT-α | Sigma (P4359) |
| SB431542 | Tocris (1614) |
| VPA | Tocris (2815) |
| TTNPB | Tocris (0761) |
| 3-DZNep | Sigma (5.06069) |
| UNC0379 | Cayman (16400) |
| 5-AzaC | Tocris (3842) |
| ABT-869 | Tocris (7743) |
| 2-PCPA | Tocris (3852) |
| RepSox | Tocris (3742) |
| Compound E | Tocris (6476) |
| DEX | Tocris (1126) |
| HH | Tocris (4093) |
| Paraformaldehyde (Paraformaldehyde) | Sigma (158127) |
| DAPI | Sigma (D9542) |
| Normal Donkey Serum (Normal Donkey Serum) | Jackson Lab (017-000-12) |
| Alkaline Phosphatase Detection Kit (Alkaline Phosphatase Detection Kit) | Sigma (SCR004) |
| Hematoxylin and Eosin Stain Kit (Hematoxylin and Eosin staining Kit) | Abcam (H-3502) |
| Evo M-MLV RT Kit with gDNA Clean for qPCR (in vitro reverse transcription Kit) | AG (AG11705) |
| SYBR Green Premix Pro Taq HS qPCR Kit (SYBR Green Premix Pro Taq HS qPCR Kit) | AG (AG11718) |
| Trizol | Invitrogen (15596018) |
| Demecolcine (colchicine) | Sigma (D7385) |
TABLE 4 reagent names
| SB431542 | TGF-beta signaling receptor inhibitors |
| VPA | Histone deacetylase inhibitors |
| TTNPB | RAR signaling pathway agonists |
| 3-DZNEP | Histone methyltransferase inhibitors |
| UNC0379 | Methyltransferase SETD8 inhibitors |
| 5-Aza | DNA methyltransferase inhibitors |
| Linifanib | VEGFR/PDGFR inhibitors |
| 2-PCPA | LSD1 inhibitors |
| RepSox | TGF-beta signaling receptor inhibitors |
| CHIR99021 | GSK-3 inhibitors |
| GlutaMax | Glutamine |
| NEAA | Non-essential amino acids |
| FBS | Fetal bovine serum |
| DMEM-F12 | Dulbecco's Modified Eagle mammalian cell culture medium |
| PFT-α | P53 inhibitors |
| Y-27632 | ROCK signal pathway inhibitors |
| NaB | Pyruvic acid sodium salt |
| PD0325901 | Inhibitors of MEK signaling pathways |
| SCF | Platelet derived growth proteins |
| IL-3 | Interleukin-3 |
| EPO | Erythropoietin |
| IGF-1 | Insulin-like growth factor |
| dexamethasone | Dexamethasone |
| 1-thioglycerol | 1-thioglycerol |
| RPMI1640 | Roswell Park mental Institute mammalian growth medium |
| B27 | Serum substitute |
TABLE 5 list of antibodies
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, which is intended to cover any modifications, equivalents, improvements, etc. within the spirit and scope of the present invention.
Claims (9)
1. A method of promoting reprogramming of somatic cells to induced pluripotent stem cells, comprising: the method comprises the following steps:
1) Introducing a plasmid containing a reprogramming factor into a somatic cell by an electroporation method;
2) Culturing in culture medium containing additives TTNPB, 2-PCPA and Repsox for 7-8 days;
3) The culture is continued for 6-7 days in a medium containing additives TTNPB, 2-PCPA and UNC0379.
2. The method of promoting reprogramming of somatic cells to induced pluripotent stem cells of claim 1, wherein the method comprises: TTNPB is 0.5-5 μ M,2-PCPA is 10-20 μ M, repsox is 0.5-5 μ M, and UNC0379 is 1-5 μ M.
3. The method of promoting reprogramming of somatic cells to induced pluripotent stem cells of claim 1, wherein the method comprises: the medium of step 2) comprises a base medium consisting of 1% GlutaMax, 1% NEAA, 10% FBS DMEM-F12, and additives comprising 1-5. Mu.M CHIR99021, 0.5-3. Mu.M PFT-alpha, 5-15. Mu.M Y-27632, 100-500. Mu.M NaB.
4. The method of promoting reprogramming of somatic cells to induced pluripotent stem cells of claim 1, wherein the method comprises: the culture medium of the step 3) comprises a basic culture medium and additives, wherein the basic culture medium is mTeSR1, and the additives further comprise 1-5 mu M CHIR99021, 0.5-3 mu M PFT-alpha, 10-15 mu M Y-27632, 100-500 mu M NaB and 0.1-1 mu M PD0325901.
5. The method of promoting reprogramming of somatic cells to induced pluripotent stem cells according to claim 1, wherein: reprogramming factors include OCT4, SOX2, KLF4, and C-Myc.
6. The method of promoting reprogramming of somatic cells to induced pluripotent stem cells according to claim 5, wherein: the plasmids are 2-5. Mu.g of PCEP4-OCT4-IRES-SOX2 plasmid, 0.5-2. Mu.g of PCEP4-KLF4 plasmid and 0.5-2. Mu.g of PCEP4-C-Myc plasmid.
7. A culture medium for use in the method of any one of claims 1 to 6, characterized in that: the culture medium comprises a culture medium A and a culture medium B, wherein the culture medium A comprises additives TTNPB, 2-PCPA and Repsox, and the culture medium B comprises additives TTNPB, 2-PCPA and UNC0379.
8. An induced pluripotent stem cell constructed by the method of any one of claims 1 to 6.
9. Use of the induced pluripotent stem cell of claim 8 for establishing a disease model.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310101650.4A CN115772505B (en) | 2023-02-13 | 2023-02-13 | Culture medium and method for promoting reprogramming of somatic cells into induced pluripotent stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310101650.4A CN115772505B (en) | 2023-02-13 | 2023-02-13 | Culture medium and method for promoting reprogramming of somatic cells into induced pluripotent stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN115772505A true CN115772505A (en) | 2023-03-10 |
| CN115772505B CN115772505B (en) | 2023-05-19 |
Family
ID=85393578
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310101650.4A Active CN115772505B (en) | 2023-02-13 | 2023-02-13 | Culture medium and method for promoting reprogramming of somatic cells into induced pluripotent stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115772505B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116515742A (en) * | 2023-07-04 | 2023-08-01 | 夏同生物科技(苏州)有限公司 | Method for reprogramming human cells into induced pluripotent stem cells |
| CN116536255A (en) * | 2023-07-05 | 2023-08-04 | 夏同生物科技(苏州)有限公司 | Culture medium and method for efficiently inducing muscle stem cells |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110218696A (en) * | 2018-03-01 | 2019-09-10 | 中国科学院广州生物医药与健康研究院 | A kind of cultivating system generated for chemical induction multipotent stem cells and the chemical reprogramming method using the cultivating system |
| CN112831462A (en) * | 2021-02-09 | 2021-05-25 | 复旦大学 | Compositions, media and methods for inducing reprogramming of human cells to induced pluripotent stem cells |
| CN113454230A (en) * | 2019-08-01 | 2021-09-28 | 浙江霍德生物工程有限公司 | Method for preparing induced pluripotent stem cells through somatic cell reprogramming |
| CN113881637A (en) * | 2021-08-30 | 2022-01-04 | 山西医科大学 | Human induced pluripotent stem cell and preparation method and application thereof |
| CN114645023A (en) * | 2022-05-18 | 2022-06-21 | 上海爱萨尔生物科技有限公司 | System and method for reprogramming peripheral blood mononuclear cells to induced pluripotent stem cells |
| WO2022213731A1 (en) * | 2021-04-08 | 2022-10-13 | Peking University | Chemical reprogramming of human somatic cells into pluripotent cells |
-
2023
- 2023-02-13 CN CN202310101650.4A patent/CN115772505B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110218696A (en) * | 2018-03-01 | 2019-09-10 | 中国科学院广州生物医药与健康研究院 | A kind of cultivating system generated for chemical induction multipotent stem cells and the chemical reprogramming method using the cultivating system |
| CN113454230A (en) * | 2019-08-01 | 2021-09-28 | 浙江霍德生物工程有限公司 | Method for preparing induced pluripotent stem cells through somatic cell reprogramming |
| CN112831462A (en) * | 2021-02-09 | 2021-05-25 | 复旦大学 | Compositions, media and methods for inducing reprogramming of human cells to induced pluripotent stem cells |
| WO2022213731A1 (en) * | 2021-04-08 | 2022-10-13 | Peking University | Chemical reprogramming of human somatic cells into pluripotent cells |
| CN113881637A (en) * | 2021-08-30 | 2022-01-04 | 山西医科大学 | Human induced pluripotent stem cell and preparation method and application thereof |
| CN114645023A (en) * | 2022-05-18 | 2022-06-21 | 上海爱萨尔生物科技有限公司 | System and method for reprogramming peripheral blood mononuclear cells to induced pluripotent stem cells |
Non-Patent Citations (1)
| Title |
|---|
| JINGYANG GUAN等: "Chemical reprogramming of human somatic cells to pluripotent stem cells" * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116515742A (en) * | 2023-07-04 | 2023-08-01 | 夏同生物科技(苏州)有限公司 | Method for reprogramming human cells into induced pluripotent stem cells |
| CN116515742B (en) * | 2023-07-04 | 2023-09-01 | 夏同生物科技(苏州)有限公司 | Method for reprogramming human cells into induced pluripotent stem cells |
| CN116536255A (en) * | 2023-07-05 | 2023-08-04 | 夏同生物科技(苏州)有限公司 | Culture medium and method for efficiently inducing muscle stem cells |
| CN116536255B (en) * | 2023-07-05 | 2023-09-12 | 夏同生物科技(苏州)有限公司 | Culture medium and method for efficiently inducing muscle stem cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN115772505B (en) | 2023-05-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Karlsson et al. | Human embryonic stem cell-derived mesenchymal progenitors—potential in regenerative medicine | |
| CN115772505B (en) | Culture medium and method for promoting reprogramming of somatic cells into induced pluripotent stem cells | |
| Ishkitiev et al. | High-purity hepatic lineage differentiated from dental pulp stem cells in serum-free medium | |
| EP1384775B1 (en) | Somatic pluripotent cells | |
| Larijani et al. | Retracted: Long-Term Maintenance of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells in Suspension | |
| JP2017140052A (en) | Creation of myocardial cell | |
| KR20080056181A (en) | Method of deriving progenitor cell line | |
| US9181529B2 (en) | Titration of differentiation medium components | |
| US20070298016A1 (en) | Methods For The Generation Of Hepatocyte-Like Cells From Human Blastocyst-Derived Stem (Hbs) | |
| US8507275B2 (en) | Method of inducing differentiation of embryonic stem cells into hemangioblast | |
| CN110713973B (en) | Culture medium combination and method for inducing pluripotent stem cells to differentiate into mesenchymal stem cells | |
| JP6860539B2 (en) | Adhesive signature-based method for isolating stem cells and cells derived from stem cells | |
| EP3207122A1 (en) | Generation of keratinocytes from pluripotent stem cells and maintenance of keratinocyte cultures | |
| US20200140826A1 (en) | Maintenance and Expansion of Pancreatic Progenitor Cells | |
| CN105907705B (en) | Pluripotent stem cell culture medium | |
| WO2019109666A1 (en) | Method for culturing urine-derived cells | |
| JP7209619B2 (en) | Non-human primate induced pluripotent stem cell-derived hepatocytes and uses thereof | |
| Inanç et al. | Differentiation of human embryonic stem cells on periodontal ligament fibroblasts in vitro | |
| EP2134836B1 (en) | A combined scalable in vitro differentiation system for human embryonic stem cells for direct assay application in multiwell plates | |
| AU2007228982B2 (en) | Culture system and method for propagation of human blastocyst-derived stem cells | |
| KR20150044691A (en) | Method for inducing differentiation of human pluripotent stem cells into nephron progenitor cells | |
| Keil et al. | Transcriptional expression profile of cultured human embryonic stem cells in vitro and in vivo | |
| KR102218549B1 (en) | Human salivary gland cell culture method | |
| US20230078230A1 (en) | Methods for the production of committed cardiac progenitor cells | |
| CN117402814A (en) | Endothelial induction culture medium and method for inducing differentiation of pluripotent stem cells into vascular endothelial cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |