CN112941019B - Method for promoting reprogramming of somatic cells - Google Patents

Method for promoting reprogramming of somatic cells Download PDF

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CN112941019B
CN112941019B CN202110291290.XA CN202110291290A CN112941019B CN 112941019 B CN112941019 B CN 112941019B CN 202110291290 A CN202110291290 A CN 202110291290A CN 112941019 B CN112941019 B CN 112941019B
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CN112941019A (en
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祝赛勇
王卫云
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Zhejiang University ZJU
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Abstract

The invention discloses a method for promoting somatic cell reprogramming, namely, a method for promoting somatic cell reprogrammingMethods for exogenous addition or endogenous regulation to increase H in somatic cells 2 S is generated, and reprogramming of somatic cells is further promoted. Wherein the exogenous addition is a chemical induction method, namely adding N-acetyl-L-cysteine into a basal culture medium to obtain a reprogramming culture medium; culturing somatic cells in the early reprogramming stage by using a reprogramming culture medium; by increasing H in somatic cells 2 Production of S ultimately promotes reprogramming of somatic cells. Endogenous regulation is a gene over-expression method, namely, a somatic cell to be reprogrammed is transfected with a gene over-expression virus, so that the somatic cell over-expresses a Cbs gene; by increasing H in somatic cells 2 Production of S ultimately promotes reprogramming of somatic cells. The method can obviously improve the efficiency of somatic cell reprogramming and promote the application of Induced Pluripotent Stem Cells (iPSCs).

Description

Method for promoting reprogramming of somatic cells
Technical Field
The invention belongs to somatic cell reprogramming Cheng Lingyu, and particularly relates to a method for promoting somatic cell reprogramming.
Background
Stem cells can now be obtained in several ways:
1) Isolated from the blastocyst. Embryonic Stem Cells (ESCs) are derived from the inner cell mass of the blastocyst stage of Embryonic development, are pluripotent and can proliferate indefinitely, and are powerful tools for studying self-renewal, development and disease. However, the acquisition of embryonic stem cells involves ethical problems and there are immunological rejection problems in clinical applications.
2) And (4) nuclear transplantation. The transfer of somatic cell nuclei into enucleated oocytes eliminates the somatic cell characteristics and has the potential to develop into intact individuals. However, this technique is difficult to operate and involves ethical problems.
3) And (4) fusing the cells. The hybrid cell obtained by fusing the somatic cell and the stem cell can eliminate the characteristics of the somatic cell and obtain the biochemical and developmental characteristics of the pluripotent cell. Can be used to study genetic complementation and search for factors that reprogram somatic cells. However, the cells obtained by this method are not normal chromosome cells and cannot be used for clinical applications.
4) Transcription factor-induced somatic reprogramming. Reprogramming refers to restoring already differentiated cells to the state of stem cells having differentiation ability by a certain means. The somatic cells are reprogrammed by transferring transcription factors (Oct 4, sox2, klf4 and c-Myc) into the somatic cells to induce the production of pluripotent stem cells (iPSCs). The cells obtained by the method can be derived from patients, so that immunological rejection does not occur in clinical application, and ethical problems are not involved. However, the genome of the cell integrates exogenous genes during reprogramming, and the clinical application has the risk of tumor formation.
5) Small molecule induced somatic reprogramming (chemical reprogramming). When the somatic cells are cultured, the small molecular compound is added to induce the somatic cells into pluripotent stem cells. The stem cells obtained by the method can be derived from somatic cells of patients, so that immune rejection and ethical problems are eliminated; and no foreign gene is inserted, so that the clinical application is safer. However, the method is low in efficiency, long in time consumption and unclear in molecular mechanism at present, and clinical application of stem cells is limited.
Therefore, the search for new small molecules to improve reprogramming efficiency and reveal mechanisms therein is very necessary for the clinical application of iPSCs.
Disclosure of Invention
It is an object of the present invention to overcome the disadvantages of the prior art and to provide a method for promoting reprogramming of somatic cells.
The invention adopts the following specific technical scheme:
the invention provides a method for promoting somatic cell reprogramming, namely a method for improving H in somatic cells by exogenous addition or endogenous regulation 2 Production of S, in turn, promoted reprogramming of somatic cells (denoted as protocol A).
Preferably, the somatic cells are mouse embryonic fibroblasts (protocol B).
Preferably, in either of embodiments a and B, the exogenous addition is a chemical induction method.
Further, the chemical induction method is as follows:
adding N-acetyl-L-cysteine into the basic culture medium to obtain a reprogramming culture medium; culturing cells in an early reprogramming stage by using a reprogramming culture medium; by increasing H in somatic cells 2 Production of S fromAnd the activity of mitochondrial oxidative phosphorylation and intracellular redox homeostasis are regulated, and reprogramming of somatic cells is finally promoted.
Further, the concentration of N-acetyl-L-cysteine in the reprogramming medium is 5mM.
Furthermore, the initial concentration of N-acetyl-L-cysteine is 1M, and the mixing ratio of N-acetyl-L-cysteine to the basic medium is 1.
Wherein, the first chemical induction method comprises the following steps:
1) Inoculating somatic cells to be reprogrammed in MEF medium;
2) After culturing the somatic cells in MEF medium for 1 day, the MEF medium is changed to the first reprogramming medium; the first reprogramming culture medium is Stage 1 culture medium added with NAC; the Stage 1 medium comprises 78% DMEM,10% KSR,10% FBS,1% NEAA,1% P/S,0.055mM β -ME,20ng/mL bFGF,0.5mM VPA,20 μ M CHIR99021, 10 μ M616452,5 μ M Parnate,50 μ M Forskolin,0.5 μ M AM580,5 μ M EPZ004777 and 250 μ M vitamin C;
3) Replacing the first reprogramming culture medium once every 4 days, and continuously culturing for 12 days;
4) On day 12 of somatic cell culture reprogramming, replacing the first reprogramming media with Stage 2 media; the Stage 2 medium contained 78% DMEM,10% KSR,10% FBS,1% NEAA,1% P/S,0.055mM β -ME,20ng/mL bFGF,0.5mM VPA,10 μ M CHIR99021, 10 μ M616452,5 μ M Parnate,10 μ M Forskolin,0.5 μ M AM580,0.05 μ M Dep ZN, 0.5 μ M5-aza-dC, 5 μ M SGC0946 and 250 μ M Vc;
5) Replacing the Stage 2 culture medium once every 4 days, and continuously culturing for 12 days;
6) On day 24 of somatic cell culture reprogramming, replacing the Stage 2 culture with Stage 3 culture medium; the Stage 3 medium comprises 47% DMEM/F12, 47% neurobasal medium, 1 XN 2 supplement,1 XB 27 supplement,1% NEAA,1% P/S,0.055mM beta-ME, 1,000U/mL LIF, 3. Mu.M CHIR99021 and 0.2. Mu.M PD0325901;
7) And replacing the fresh culture medium of the Stage 3 every 4 days, continuously culturing for 12-16 days, and finishing the reprogramming culture process of the adult cells.
The second chemical induction method specifically comprises the following steps:
1) Subjecting somatic cells to be reprogrammed to lentiviral transfection, the lentiviruses expressing Oct4, sox2, klf4 and c-Myc genes;
2) Subculturing the transfected somatic cells;
3) Placing the somatic cells after subculture in a second reprogramming culture medium, replacing the second reprogramming culture medium once every 4 days, and continuously culturing for 8 days to finish the reprogramming culture process of the somatic cells; the second reprogramming medium is an initial medium added with NAC; the initial medium comprises 78% DMEM,10% KSR,10% FBS,1% NEAA,1% P/S,0.055mM β -ME and 1,000U/mL mLIF,2 μ g/mL doxycycline.
Preferably, the first and second chemical induction methods are performed in a first reprogramming media and a second reprogramming media each at a NAC concentration of 5mM.
Preferably, in case of variant A or B, the endogenous regulation is a gene overexpression process.
Further, the gene overexpression method comprises the following steps:
transfecting somatic cells to be reprogrammed with a gene over-expression virus so that the somatic cells over-express the Cbs gene; by increasing H in somatic cells 2 And S is generated, so that the mitochondrial oxidative phosphorylation activity and the intracellular redox homeostasis are regulated, and the reprogramming of somatic cells is finally promoted.
Wherein, the steps of the gene overexpression method are as follows:
1) Amplifying CDS segments of the Cbs genes from a cDNA library, and then connecting the CDS segments with large segments of an overexpression vector to obtain an overexpression plasmid;
2) Packaging the over-expression plasmid into a gene over-expression virus;
3) Adding somatic cells to be reprogrammed into a culture plate with a culture medium, removing the culture medium in the culture plate on the next day of reprogramming, adding the packaged viruses into the culture plate, and replacing the packaged viruses with a fresh culture medium after 4 hours to ensure that the Cbs genes of the somatic cells begin to be over-expressed;
4) On day 12 of somatic cell reprogramming, cells were subjected to Sall4 immunofluorescence staining, and clones were counted to calculate reprogramming efficiency.
Compared with the prior art, the invention has the following beneficial effects:
1) The method can obviously improve the efficiency of somatic cell reprogramming and promote the application of Induced Pluripotent Stem Cells (iPSCs).
2) The method of the invention can increase intracellular H 2 S level, H accumulation in somatic cells 2 S can regulate and control the activity of mitochondrial oxidative phosphorylation and intracellular redox homeostasis, and finally promotes somatic cell reprogramming.
Drawings
FIG. 1 is a graph of HA and NAC vs H 2 S effects are compared to graphs in which HA:250 μ M, NAC:5mM, DPBS as a control group.
FIG. 2 is a graph of the effect of HA on chemical reprogramming.
FIG. 3 is a graph of the effect of NAC on chemical reprogramming.
FIG. 4 is a graph of the effect of HA on transcription factor-induced cell reprogramming.
FIG. 5 is a graph of the effect of NAC on transcription factor-induced cell reprogramming.
Fig. 6 shows Cbs gene knock-out efficiency assay results, where ShNC is the control group.
Fig. 7 shows the detection result of the Cth gene knockdown efficiency, wherein ShNC is a control group.
FIG. 8 is a graph of the effect of Cbs gene knock-down on somatic cell reprogramming.
FIG. 9 is a graph showing the effect of Cth gene knock-down on reprogramming of somatic cells.
FIG. 10 is a graph showing the effect of overexpression of the Cbs gene on reprogramming somatic cells, in which pSin-GFP was used N Is a control group.
Detailed Description
The invention will be further elucidated and described with reference to the drawings and the detailed description. The technical features of the embodiments of the present invention can be combined correspondingly without mutual conflict.
The invention provides a method for promoting somatic cell reprogramming, which is used for increasing H in somatic cells by means of exogenous addition or endogenous regulation 2 S is generated, and reprogramming of somatic cells is further promoted. The method of the present invention can be applied to reprogramming all somatic cells including mouse embryonic fibroblasts, and can effectively promote the process thereof. The method will be specifically described below.
The exogenous addition means specifically refers to a chemical induction method, namely adding N-acetyl-L-cysteine (NAC) into a basal culture medium to obtain a reprogramming culture medium; culturing somatic cells in the early reprogramming stage by using a reprogramming culture medium; by increasing H in somatic cells 2 And S is generated, so that the mitochondrial oxidative phosphorylation activity and intracellular redox homeostasis are regulated, and the reprogramming of somatic cells is finally promoted.
The NAC concentration in the reprogramming media can be 5mM, and in order to achieve this concentration of NAC in the reprogramming media, the initial concentration of NAC can be set to 1m with a mix ratio of NAC to basal media of 1.
The endogenous regulation means specifically refers to a gene overexpression method. Transfecting a somatic cell to be reprogrammed with a gene over-expression virus to ensure that the somatic cell gene over-expresses a Cbs gene; by increasing H in somatic cells 2 And S is generated, so that the mitochondrial oxidative phosphorylation activity and the cell redox homeostasis are regulated, and the reprogramming of somatic cells is finally promoted.
Example 1
The embodiment provides a chemical induction method, which comprises the following steps:
1) Inoculation of 4X 10 in one plate 5 MEFs cells were cultured overnight in MEF medium.
2) After culturing the somatic cells in MEF medium for 1 day, the MEF medium is changed to the first reprogramming medium; the first reprogramming culture medium is Stage 1 culture medium added with NAC; stage 1 medium comprises 78% DMEM,10% KSR,10% FBS,1% NEAA,1% P/S,0.055mM β -ME,20ng/mL bFGF,0.5mM VPA,20 μ M CHIR99021, 10 μ M616452,5 μ M Parnate,50 μ M Forskolin,0.5 μ M AM580,5 μ M EPZ004777 and 250 μ M vitamin C; NAC was present in the first reprogramming media at a concentration of 5mM each.
3) Replacing a new first reprogramming culture medium every 4 days, and continuously culturing for 12 days;
4) On day 12 of somatic cell culture reprogramming, the first reprogramming media was changed to Stage 2 media; stage 2 medium contained 78% DMEM,10% KSR,10% FBS,1% NEAA,1% P/S,0.055mM β -ME,20ng/mL bFGF,0.5mM VPA,10 μ M CHIR99021, 10 μ M616452,5 μ M Parnate,10 μ M Forskolin,0.5 μ M AM580,0.05 μ M Dep, 0.5 μ M5-aza-ZN dC,5 μ M SGC0946 and 250 μ M Vc;
5) Replacing the culture medium with new Stage 2 every 4 days, and continuously culturing for 12 days;
6) On day 24 of reprogramming of somatic cell cultures, stage 2 cultures were changed to Stage 3 medium; stage 3 medium comprising 47% DMEM/F12, 47% neurobasal medium, 1 XN 2 supplement,1 XB 27 supplement,1% NEAA,1% P/S,0.055mM β -ME,1,000U/mL LIF,3 μ M CHIR99021 and 0.2 μ M PD0325901;
7) Replacing the culture medium of Stage 3 once every 4 days, continuously culturing for 12-16 days, and finishing the reprogramming culture process of the adult cells.
Example 2
This example provides another induction method, which comprises the following steps:
1) MEFs cells were seeded in 6-well plates at a density of 60% -80% and were transfected with lentiviruses (expressing Oct4, sox2, klf4 and c-Myc genes) once a day;
2) 24 hours after the second transfection, cells were passaged into 24-well plates at 3X 10 cells per plate 5 A cell;
3) After overnight culture of the cells, placing the somatic cells after subculture in a second reprogramming culture medium, replacing the new second reprogramming culture medium every 4 days, and continuously culturing for 8 days to finish the reprogramming culture process of the somatic cells; the second reprogramming media is initial media supplemented with NAC; the starting medium comprises 78% DMEM,10% KSR,10% FBS,1% NEAA,1% P/S,0.055mM β -ME and 1,000U/mL mLIF,2 μ g/mL doxycycline. NAC was present in the second reprogramming media at a concentration of 5mM.
Example 3
This example provides a gene overexpression method, comprising the following steps:
1) Amplifying CDS fragments of the genes from a cDNA library, and then connecting the CDS fragments with an overexpression large fragment to obtain an overexpression plasmid;
2) Packaging the plasmid into a gene overexpression virus;
3) Adding somatic cells to be reprogrammed into a culture plate with a culture medium, removing the culture medium in the culture plate on the next day of reprogramming, adding the packaged virus into the culture plate, and replacing the packaged virus with a fresh culture medium after 4 hours to ensure that the Cbs gene of the somatic cells starts to be over-expressed;
4) On day 12 of somatic cell reprogramming, cells were subjected to Sall4 immunofluorescent staining, and clones were counted to calculate reprogramming efficiency.
Example 4
To further verify that NAC supplemented reprogramming media was by increasing intracellular H 2 S to effect cell reprogramming, the following comparative experiment was performed using the method of example 1:
HA and NAC were used separately at Stage 1 of chemical reprogramming, with a concentration of 250 μ M for HA (added to the medium for use at an initial concentration of 250mM, hence a ratio of 1 to 1000) and a concentration of 5mM for NAC (added to the medium at an initial concentration of 1M, hence a ratio of 1 to 200. Reprogramming day 12 intracellular H detection 2 S is changed, and the result shows that HA inhibits H 2 S production and NAC promotion effect (fig. 1). Next we tested the effect of HA and NAC on reprogramming, with Sall4 immunofluorescence staining and counting at day 12, and the results showed that 250 μ M HA significantly inhibited the efficiency of reprogramming (fig. 2), while 5mM NAC had a significant promoting effect (fig. 3).
Subsequent experiments found that the same effect was observed with HA and NAC treatment in transcription factor induced reprogramming using the method of example 2 (fig. 4 and 5).
This part of the results thus indicates intracellular H 2 The S content can be regulated and controlled by chemical reprogramming.
Example 5
To further verify that somatic cells overexpress the Cbs gene by increasing intracellular H 2 S to effect cell reprogramming, the following comparative experiment was performed using the method of example 3:
shRNA (shCbs, shCth) is designed for the genes Cbs and Cth, transfection is carried out on fibroblasts after the shRNA is packaged into viruses, and the knocking efficiency is detected, wherein the knocking efficiency of the genes Cbs and Cth is about 60 percent (figures 6 and 7). Using the method of example 1, chemical reprogramming was performed to transfect the gene-knocked-down virus on day 2, and Sall4 immunofluorescence staining and counting were performed on day 12, showing that knocking-down of both genes significantly inhibited reprogramming (fig. 8 and 9). In addition, we overexpressed the Cbs gene (pSin-Cbs). CDS segments of the genes are amplified from a cDNA library, then are connected with large segments of overexpression vectors to obtain overexpression plasmids, and the constructed plasmids are packaged into viruses, namely the overexpression viruses. Transfection of fibroblasts with an overexpressing virus, and detection of gene overexpression efficiency, we detected that Cbs were overexpressed nearly 2000-fold (probably due to underexpression of Cbs endogenous to the fibroblasts). Using the method of example 3, the gene-overexpressing virus was transfected the next day of reprogramming, and Sall4 immunofluorescence staining and counting were performed on day 12, showing that Cbs gene overexpression promotes reprogramming (FIG. 10).
Thus, these data indicate an increase in H 2 The content of S can facilitate cell reprogramming. That is, the methods of the invention are capable of increasing intracellular H 2 S level, H accumulation in somatic cells 2 S can regulate and control the activity of mitochondrial oxidative phosphorylation and intracellular redox homeostasis, and finally promotes somatic cell reprogramming.
The above embodiments are merely preferred embodiments of the present invention, which should not be construed as limiting the invention. Various changes and modifications may be made by one of ordinary skill in the pertinent art without departing from the spirit and scope of the present invention. Therefore, the technical scheme obtained by adopting the mode of equivalent replacement or equivalent transformation is within the protection scope of the invention.

Claims (4)

1. A method for promoting in vitro chemical reprogramming of somatic cells into pluripotent stem cells, comprising increasing H in somatic cells by a chemical induction method 2 S generation, and further promoting chemical reprogramming of somatic cells into pluripotent stem cells in vitro; the chemical induction method comprises the steps of adding N-acetyl-L-cysteine into a basic culture medium to obtain a reprogramming culture medium, and culturing somatic cells at the early stage of reprogramming by using the reprogramming culture medium.
2. The method of claim 1, wherein the somatic cell is a mouse embryonic fibroblast.
3. The method of claim 1, wherein the reprogramming of somatic cells to pluripotent stem cells in vitro is performed at a concentration of N-acetyl-L-cysteine of 5mM in the reprogramming media.
4. The method of claim 1, wherein the initial concentration of N-acetyl-L-cysteine is 1m and the mixing ratio of N-acetyl-L-cysteine to basal medium is 1.
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