Transcription factor composition and application thereof in reprogramming somatic cells into induced pluripotent stem cells

Abstract

The invention relates to the technical field of biology, in particular to a transcription factor composition and application thereof in reprogramming somatic cells into induced pluripotent stem cells. The transcription factor composition provided by the invention only needs the OCT4 transcription factor, the c-MYC transcription factor and the KDM8 transcription factor, overcomes the defect that the effect of reprogramming somatic cells into induced pluripotent stem cells can be realized by at least containing four transcription factors in the prior art, can realize the same effect by only needing three transcription factors, reduces the unstable factors of a genome, and has the advantages of high specificity, high sensitivity and the like. Meanwhile, the induction efficiency of inducing the pluripotent stem cells is also improved. Experiments show that compared with a classical combination of OCT4, SOX2, KLF4 and c-MYC, the transcription factor composition disclosed by the invention has the advantage that the efficiency of reprogramming human skin fibroblasts into induced pluripotent stem cells can be improved by about 26%.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a transcription factor composition and an application thereof in reprogramming somatic cells into induced pluripotent stem cells. Background Art

[0002] Induced pluripotent stem cells (iPSCs) are a type of cell that reprograms somatic cells into a dedifferentiated state through the expression of specific genes. Induced pluripotent stem cells have pluripotency similar to that of embryonic stem cells (ESCs) and can differentiate into almost all types of cells in the body. They have broad application prospects in regenerative medicine, disease modeling, and drug development. However, the lentiviral induction method for obtaining iPSCs involves the insertion of multiple factors and has a low induction efficiency, which has hindered its application to a certain extent.

[0003] In 2007, the Yamanaka research group transferred four transcription factors, OCT4, SOX2, KLF4, and c-MYC, into human fibroblasts and obtained human iPSCs for the first time. These four transcription factors (OCT4, SOX2, KLF4, c-MYC, i.e., OSKM) are generally called Yamanaka factors. This combination can successfully reprogram almost all types of cells, but the insertion of four transcription factors may cause reduced genome stability and low efficiency in inducing the generation of iPSCs. So far, although different methods of inducing pluripotent stem cells have been studied, OSKM induction is still the most classic method. There are also new methods that try to add transcription factors (such as KDM1B) on the basis of OSKM to improve the induction efficiency, but these methods all require the insertion of at least four transcription factors, which may cause reduced genome stability, unoptimized genome stability, and low efficiency in inducing the generation of iPSCs. Summary of the invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a transcription factor composition and its application in reprogramming somatic cells into induced pluripotent stem cells. The present invention provides only three transcription factors, namely, OCT4 transcription factor, c-MYC transcription factor and KDM8 transcription factor, which can improve the induction efficiency of induced pluripotent stem cells while improving genome stability.

[0005] The present invention provides a transcription factor composition, comprising an OCT4 transcription factor, a c-MYC transcription factor and a KDM8 transcription factor.

[0006] Compared with the prior art, the transcription factor composition provided by the present invention reduces the number of transcription factors, thereby reducing genomic instability factors while improving the induction efficiency to a certain extent, thereby obtaining a more accurate technical effect.

[0007] In some embodiments, in the composition, the expression ratio of OCT4 transcription factor, c-MYC transcription factor and KDM8 transcription factor is 1:(0.5~2):(0.1~1).

[0008] In some specific embodiments, in the composition, the expression ratio of OCT4 transcription factor, c-MYC transcription factor and KDM8 transcription factor is 1:1:1.

[0009] The present invention provides nucleic acid molecules, including nucleic acid molecules encoding OCT4 transcription factor, c-MYC transcription factor and KDM8 transcription factor in the transcription factor composition.

[0010] The present invention provides an expression vector comprising the nucleic acid molecule.

[0011] In some embodiments, the expression vector comprises any one or more of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, and a retroviral vector.

[0012] In some specific embodiments, the expression vector is a lentiviral vector.

[0013] The present invention provides a host cell transformed or transfected with the expression vector.

[0014] The present invention provides the use of at least one of the following (1) to (4) in reprogramming somatic cells into induced pluripotent stem cells:

[0015] (1) The transcription factor composition;

[0016] (2) the nucleic acid molecule;

[0017] (3) the expression vector;

[0018] (4) The host cell.

[0019] The present invention provides a reagent for reprogramming somatic cells into induced pluripotent stem cells, comprising at least one of ① to ④:

[0020] 1. the transcription factor composition;

[0021] ②, the nucleic acid molecule;

[0022] ③, the expression vector;

[0023] ④. The host cell.

[0024] The present invention provides a method for reprogramming somatic cells into induced pluripotent stem cells, comprising taking somatic cells, mixing them with the reagent, and culturing them to obtain the induced pluripotent stem cells.

[0025] In some embodiments, the somatic cells include any one or more of fibroblasts, peripheral blood mononuclear cells, epithelial cells, and adipocytes.

[0026] In some specific embodiments, the somatic cell is a fibroblast, specifically a human skin fibroblast.

[0027] The present invention provides induced pluripotent stem cells prepared by the method.

[0028] The present invention provides the use of the induced pluripotent stem cells in preparing products for regenerative medicine, disease modeling and drug development.

[0029] The present invention provides a product, comprising the induced pluripotent stem cells.

[0030] Compared with the prior art, the transcription factor composition provided by the present invention only requires OCT4 transcription factor, c-MYC transcription factor and KDM8 transcription factor, which overcomes the prior art that at least four transcription factors are included to achieve the effect of reprogramming somatic cells into induced pluripotent stem cells. The present invention only requires three transcription factors to achieve the same effect, reducing the genomic instability factor and improving the induction efficiency of induced pluripotent stem cells. Experiments show that the efficiency of reprogramming human skin fibroblasts into induced pluripotent stem cells can be increased by about 26% using the transcription factor composition of the present invention compared with the classic OCT4, SOX2, KLF4, c-MYC combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The figure shows the result of using alkaline phosphatase staining of two groups of induced cells in Example 1;

[0032] Figure 2 Statistical results of alkaline phosphatase staining of two groups of induced cells in Example 1 are shown;

[0033] Figure 3 The figure shows the result of using alkaline phosphatase staining for two groups of induced cells in Example 2;

[0034] Figure 4 Shows the results of identification of pluripotency of iPSCs cells induced by OMK combination;

[0035] Figure 5 A diagram showing the results of in vitro three-germ layer differentiation identification of iPSCs cells induced by the OMK combination. DETAILED DESCRIPTION

[0036] The present invention provides a transcription factor composition and its use in reprogramming somatic cells into induced pluripotent stem cells. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0037] The present invention focuses on the transcription factor KDM8. Compared with the existing methods, KDM8 can replace multiple transcription factors. The combination of three transcription factors (OCT4, c-MYC, KDM8) successfully obtained iPSCs cells, reduced genomic instability factors, and improved the induction efficiency of iPSCs (about 26% higher than the classic method). The protection focus is on this transcription factor combination, and the induced cell type, induced virus dosage, and induced culture medium in other steps can be used according to actual conditions.

[0038] The present invention focuses on the combination of transcription factors OCT4, c-MYC, and KDM8 to induce iPSCs, which can not only reduce genomic instability factors, but also improve the induction efficiency to a certain extent. The experiment only takes human skin fibroblasts as an example, but since the classic Yamanaka factor combination (OSKM) can successfully reprogram all types of cells, the OCT4, c-MYC, and KDM8 combination of this method should have similar potential to reprogram other cell types besides HDF cells.

[0039] The present invention uses HDF (human skin fibroblasts) as somatic cells for reprogramming. HDF is a more common and classic cell type for inducing iPSCs among terminally differentiated somatic cells. Most of the literature uses this cell for induction (for example, human induced pluripotent stem cells were initially obtained by Shinya Yamanaka using this cell type, and another article on KDM1B in the attachment also used HDF cells for iPSCs induction-related research). In addition, the source of HDF cells is simpler than other mesenchymal stem cells, and the efficiency of induction using HDF cells is generally higher. This cell is suitable for exploring iPSCs induction methods and obtaining iPSCs.

[0040] The test materials used in the present invention are all common commercial products and can be purchased in the market. The present invention is further described below in conjunction with the embodiments.

[0041] Example 1

[0042] The method for reprogramming human skin fibroblasts into induced pluripotent stem cells (iPSCs) provided by the present invention comprises the following steps:

[0043] 1. Preparation of lentiviral vector and calculation of viral titer

[0044] Transcription factor combination design:

[0045] This experiment uses two groups of transcription factor combinations for induction:

[0046] Combination 1 is the classic OCT4, SOX2, KLF4, c-MYC, i.e., OSKM combination, and the expression ratio of each transcription factor is 1:1:1:1;

[0047] Combination 2 is a combination of OCT4, c-MYC, KDM8, or OMK, and the expression ratio of each transcription factor is 1:1:1.

[0048] Virus titer determination: The virus titer (TU / mL) of the lentiviral vector was determined by quantitative PCR or fluorescent labeling.

[0049] Calculation of virus dosage:

[0050] The induction was calculated according to the formula 10 5 Amount of virus required per HDF cell:

[0051] The volume of virus required for induction = the number of cells to be infected × MOI virus titer The volume of virus required for induction = the number of cells to be infected × MOI virus titer

[0052] The MOI (multiplicity of infection) of HDF cells is 20. For example, if the virus titer of transcription factor OCT4 is 10 8 TU / mL, then infection 10 5 The amount of virus required for each HDF cell is: (10 5 ×20)10 8 =20µL. Use this method to calculate the amount of virus required for each transcription factor combination.

[0053] 2. Cultivation and plating of human dermal fibroblasts (HDFs)

[0054] Cell culture: HDF cells were cultured in human fibroblast medium (containing 10% fetal bovine serum) at 37°C and 5% CO2.

[0055] Cell passaging and plating: When the cell confluence reached 90%, the cells were digested with 0.25% trypsin-EDTA and passaged at a ratio of 1:3. The cell suspension was inoculated in a 6-well plate with a plating density of 10 per well. 5 Continue to culture until the cells adhere to the wall and reach a suitable state for infection.

[0056] 3. Infection of HDF Cells with Lentivirus

[0057] Optimization of infection conditions: 24 hours after HDF cells were plated, the medium was removed and infection medium containing the lentivirus mixture (containing 8 µg / mL polyetheramine) was added.

[0058] Infection process: Cells were co-cultured with the virus mixture at 37°C, 5% CO2 for 12 hours, then replaced with fresh human fibroblast medium and continued to be cultured.

[0059] 4. Cell reprogramming and iPSCs induction

[0060] Cell transfer and matrigel coating: On the 4th day after infection, cells were digested with 0.25% trypsin-EDTA and the cell suspension was transferred to a 6-well plate pre-coated with stem cell matrigel.

[0061] Media Changes: 24 hours after cells attach, change the media to TeSR E8 (a feeder-free, chemically defined pluripotent stem cell culture medium) and replace with fresh media daily thereafter.

[0062] Cell clone observation: Culture in TeSR E8 medium for 20-25 days until cell clones with morphology similar to human embryonic stem cells (hES) appear.

[0063] 5. Identification of iPSCs clones and evaluation of induction efficiency

[0064] Alkaline phosphatase staining: Use an alkaline phosphatase staining kit to stain the cell clones. The staining results are as follows: Figure 1 shown.

[0065] Induction efficiency calculation: Count the number of alkaline phosphatase positive clones in each well. The statistical results of positive clones are as follows: Figure 2 shown.

[0066] Result analysis: Comparing the induction efficiency of the two transcription factor combinations (combination 1 and combination 2), the induction efficiency can be increased by about 26% compared with combination 1.

[0067] In the classic OSKM combination to induce iPSCs, generally four factors must be present at the same time to successfully induce somatic cells into iPSCs. Our method (OMK combination) replaced the OSKM of the classic system, reduced the total number of transcription factors required for induction, successfully obtained iPSCs, and also improved the induction efficiency to a certain extent.

[0068] Example 2

[0069] The induction method is the same as in Example 1. In this example, the transcription factor OM (OCT4 and c-MYC) or MK (c-MYC and KDM8) combination is used for induction. The alkaline phosphatase staining results are as follows: Figure 3 shown.

[0070] The results showed that when the transcription factor OCT4 or c-MYC induction was removed, that is, the OM (OCT4, c-MYC) combination and MK (c-MYC, KDM8) combination induction were used, the effect was poor and almost no iPSCs-like clones could be formed.

[0071] Effect example

[0072] The pluripotency of iPSCs cells induced by OMK combination was identified. The induced clones were subjected to immunofluorescence staining of NANOG, a pluripotency marker. The results are as follows: Figure 4 As shown, the results showed that the cell clones induced by the OMK combination can express the pluripotency marker gene NANOG.

[0073] The iPSCs cells were differentiated into three germ layers in vitro, and the marker genes of each germ layer were identified by immunofluorescence. Figure 5 As shown, the results of marker genes of each germ layer were all positive, that is, the obtained clones had the ability to differentiate into three germ layers.

[0074] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A transcription factor composition, characterized in that Including OCT4 transcription factor, c-MYC transcription factor and KDM8 transcription factor.

2. The transcription factor composition according to claim 1, characterized in that In the composition, the expression ratio of OCT4 transcription factor, c-MYC transcription factor and KDM8 transcription factor is 1: (0.5~2): (0.1~1).

3. A nucleic acid molecule, characterized in that It comprises nucleic acid molecules encoding the OCT4 transcription factor, c-MYC transcription factor and KDM8 transcription factor in the transcription factor composition of claim 1 or 2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. The expression vector according to claim 4, characterized in that The expression vector includes any one or more of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector and a retroviral vector.

6. A host cell transformed or transfected with the expression vector according to claim 4 or 5.

7. Use of at least one of the following (1) to (4) in reprogramming somatic cells into induced pluripotent stem cells: (1) The transcription factor composition according to claim 1 or 2; (2) The nucleic acid molecule according to claim 3; (3) The expression vector according to claim 4 or 5; (4) The host cell according to claim 6.

8. An agent for reprogramming somatic cells into induced pluripotent stem cells, characterized in that Include at least one of ①~④: ①. The transcription factor composition according to claim 1 or 2; ②, the nucleic acid molecule according to claim 3; ③. The expression vector according to claim 4 or 5; ④. The host cell according to claim 6.

9. A method for reprogramming somatic cells into induced pluripotent stem cells, characterized in that: The method comprises taking somatic cells, mixing them with the reagent according to claim 8, and culturing them to obtain the induced pluripotent stem cells.

10. The method according to claim 9, characterized in that The somatic cells include any one or more of fibroblasts, peripheral blood mononuclear cells, epithelial cells and adipocytes.

11. Induced pluripotent stem cells obtained by the method according to claim 9 or 10.

12. Use of the induced pluripotent stem cells according to claim 11 in preparing products for regenerative medicine, disease modeling and drug development.

13. A product, characterized in that Comprising the induced pluripotent stem cell according to claim 11.

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