CN110804582A

CN110804582A - Somatic cell reprogramming method and application thereof

Info

Publication number: CN110804582A
Application number: CN201911040324.7A
Authority: CN
Inventors: 刘兴国; 邬毅; 陈可实
Original assignee: Guangdong Provincial Laboratory Of Regenerative Medicine And Health
Current assignee: Guangdong Provincial Laboratory Of Regenerative Medicine And Health
Priority date: 2019-10-29
Filing date: 2019-10-29
Publication date: 2020-02-18

Abstract

The invention discloses a somatic cell reprogramming method, which comprises the following steps: preparing somatic cells of different sources to express one or more of Sox2, Klf4, Oct4 and c-Myc; preparation of a Medium for Programming: serum substitute additives, growth factors and small molecular compounds are added into a basic culture medium; then adding the compound into the basic culture medium, and reversing and mixing uniformly; and culturing the somatic cells in the programming culture medium to obtain the induced pluripotent stem cells. The method greatly improves the efficiency of reprogramming somatic cells into induced pluripotent stem cells. The method can promote the transformation of the mesenchyme of the somatic cells to the epithelium in a serum-free culture medium, thereby improving the reprogramming efficiency of the somatic cells from different sources and providing technical support for the application of induced pluripotent stem cells in regenerative medicine in the future.

Description

Somatic cell reprogramming method and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a somatic cell reprogramming method and application thereof.

Background

With the intensive stem cell research, scientists have found that terminally differentiated mammalian cells are also totipotent, adult cells can be reprogrammed, gene expression profiles altered to change their fate, re-pluripotent or transdifferentiated directly into another cell. There are two main approaches to early somatic reprogramming: nuclear transfer and cell fusion, but both methods still require involvement of the embryo and are extremely limited in ethical terms. In 2006, the japanese Yamanaka laboratory obtained a breakthrough research result in the field of reprogramming, and found that by exogenously expressing four transcription factors Sox2, Oct4, Klf4 and c-Myc, mouse fibroblasts can be reprogrammed to pluripotent stem cells (ipscs), a new direction is opened for stem cell research, and thus the nobel prize is obtained. The subsequent short-term years have seen an unusually rapid progression in the field of somatic reprogramming. Although many problems are encountered in the early stage of research on induced pluripotent stem cells, including foreign oncogene insertion, low efficiency, and even autoimmune reaction, the proposal of various improved methods has greatly promoted the research on the mechanism and application of reprogramming technology. The reprogramming somatic cell is not only a mouse fibroblast cell through the method improvement, and the reprogramming technology is applied to various species and cells. Scientists also screen various reprogramming factor combinations, which not only improves the reprogramming efficiency and quality, but also avoids the insertion of oncogenes, and also develops methods such as protein induction, small molecule compound induction and the like.

The goal of reprogramming technology is to solve the problem of large numbers of patient-specific pluripotent cells in the clinic. Scientists have successfully treated sickle anemia mice in the laboratory with reprogramming and gene repair techniques. Although this technique is still far from clinical use, scientists have successfully induced a number of patient iPS cells for single gene mutation disease for drug screening and gene repair. Reprogramming is an alteration in cell fate as well as developmental differentiation. However, it is also highly desirable to know what happens during the process of cell transformation to other cells.

However, at present, how to change cell fate, especially to facilitate reprogramming of somatic cells, remains to be further studied.

Disclosure of Invention

One of the objectives of the present invention is to provide a method for reprogramming somatic cells, which can effectively improve the efficiency of reprogramming somatic cells.

The technical scheme for achieving the purpose is as follows.

A method of somatic cell reprogramming comprising the steps of:

preparing somatic cells of different sources to express one or more of Sox2, Klf4, Oct4 and c-Myc;

preparation of a Medium for Programming: adding a serum substitute additive, a growth factor and a small molecular compound into a serum-free basic culture medium to obtain a serum-free basic culture medium; then adding the compound into the serum-free basal medium, and reversing and mixing uniformly; obtaining a prepared culture medium for programming;

and culturing the somatic cells in the programming culture medium, wherein the cells can generate mesenchymal transition to epithelial transition after about 2 days, and the induced pluripotent stem cells can be obtained after about 8-10 days.

In some of these embodiments, the serum replacement additive is N₂B27 and KSR.

In some of these embodiments, the N₂The concentration of the KSR is 0-1%, the concentration of the B27 is 0-2%, the concentration of the KSR is 0-15%, and the three are not 0 at the same time.

In some of these embodiments, the growth factor comprises at least one of bFGF and Lif and the small molecule compound comprises vitamin C.

In some of these embodiments, the concentration of bFGF is 0-10ng/ml, and the concentration of Lif is 500-2000 units/ml; more preferably, bFGF and Lif are included, wherein the concentration of the bFGF is 5ng/ml, and the concentration of the Lif is 2000 units/ml.

In some of these embodiments, the concentration of vitamin C is 10-200. mu.g/ml, more preferably, the concentration of vitamin C is 50. mu.g/ml.

In some of these embodiments, the compound is at least one of ethanolamine, CDP-ethanolamine, phosphatidylethanolamine.

In some embodiments, the ethanolamine is present at a concentration of 0-2mg/ml, the CDP-ethanolamine is present at a concentration of 0-2mg/ml, and the phosphatidylethanolamine is present at a concentration of 0-2mg/ml, wherein the three concentrations are not 0 at the same time.

In some embodiments, the ethanolamine is at a concentration of 1mg/ml, the CDP-ethanolamine is at a concentration of 1mg/ml, and the phosphatidylethanolamine is at a concentration of 1 mg/ml.

In one embodiment, the serum replacement additive is N at a concentration of 1%₂。

In some of these examples, the Basal Medium is one or a mixture of two or more of Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basic Medium Eagle (BME), F-12, RPMI1640, and α minimum Essential Medium (α MEM).

In one embodiment, the basal medium is DMEM.

In some of these embodiments, the somatic cells express one or more of Sox2, Klf4, Oct4, c-Myc.

In some of these embodiments, the somatic cells express Sox2, Klf4, and Oct4, and do not express c-Myc.

Another object of the present invention is to provide induced pluripotent stem cells prepared by the above method for reprogramming somatic cells.

The inventor of the invention adds ethanolamine, CDP-ethanolamine and phosphatidylethanolamine into one or more somatic cells expressing Sox2, Klf4, Oct4 and c-Myc from different sources in a matching way, and further combines the characteristics of preferable serum-substituting additives, growth factors, small molecular compounds and somatic cells, thereby providing the somatic cell reprogramming method. The invention adds one or more compounds to the serum-free basal medium, which can greatly improve the efficiency of reprogramming somatic cells into induced pluripotent stem cells. The method can promote the transformation of the mesenchyme of the somatic cells to the epithelium in a serum-free culture medium, thereby improving the reprogramming efficiency of the somatic cells from different sources and providing technical support for the application of induced pluripotent stem cells in regenerative medicine in the future.

Drawings

FIG. 1 is a schematic diagram showing the results of adding ethanolamine, CDP-ethanolamine and phosphatidylethanolamine in a basic serum-free culture medium containing 1% of N2 to improve the reprogramming efficiency of mouse embryonic fibroblasts and immunofluorescence and chimeric mouse experiments to identify the pluripotent stem cell clones obtained by reprogramming to have pluripotency.

FIG. 2 is a graph showing the results of increasing the reprogramming efficiency of mouse tip fibroblasts by adding ethanolamine to a basic serum-free medium containing 1% of N2 and increasing the reprogramming efficiency of mouse embryo fibroblasts by adding ethanolamine to a basic serum-free medium containing 15% of KSR.

FIG. 3 is a schematic diagram showing the results of adding ethanolamine to promote the transition of mouse fibroblast mesenchymal epithelium, promote the expression of epithelial cell marker genes, and reduce the expression of mesenchymal cell marker genes.

Detailed Description

The following examples illustrate standard laboratory practice of the inventors for illustrating the mode of the invention, and the invention should not be construed as being limited in scope to these examples. These examples are given by way of illustration only and it will be understood by those of ordinary skill in the art that various changes, modifications and adaptations may be made without departing from the scope of the invention as disclosed herein and as such are within the ordinary skill in the art. The techniques involved therein are, unless otherwise specified, conventional techniques in various fields of molecular biology, cell biology, biochemistry, and the like, which are well known to those skilled in the art.

Definitions and techniques

1. The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, immunology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and maniotis, molecular cloning, experimental guidelines, 3 rd edition (2002); current promoters IN MOLECULAR BIOLOGY (F.M. Ausubel et al (1987)); book of the series Methods IN Enzymology (Academic Press, Inc.): and (3) PCR 2: a PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor, (1995)), Harlow and Lane, Inc. (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CUTURE (R.I. Freshney, Inc. (1987)); french Anderson et al, HANDBOOK OF STEM CELLS, volume 2.

2. Unless otherwise defined, terms used herein have meanings that are conventionally understood by those skilled in the art, and some terms used herein are defined as follows in order to facilitate understanding of the present invention.

All numerical designations such as pH, temperature, time, and concentration, including ranges, are approximations. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about". It is also to be understood that, although not always explicitly recited, the reagents described herein are merely exemplary and equivalents thereof are known in the art.

The Basal media that can be used in the present invention include, but are not limited to, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium (MEM), Basic Medium Eagle (BME), F-12, RPMI1640, α minimum Essential Medium (α MEM). This is known to those skilled in the art how to select a Basal Medium suitable for the cells being cultured.

The term "serum replacement additive" as used herein refers to an additive which is added to the basal medium during cell culture to partially or completely replace serum in supporting the cell growth and survival, and generally includes factors such as insulin, transmetallic proteins, trace elements, vitamins, etc., which are not generally contained in the basal medium but are provided by serum normally used for culturing cells. The serum replacement additive comprises at least one or more of the following components that support cell growth: one or more insulins and insulin substitutes, one or more metalloproteinates and metalloproteinates substitutes, one or more trace elements, one or more vitamins, one or more amino acids, one or more hormones and hormone-like compounds, serum albumin or serum albumin substitutes, and one or more lipids, and the like. Various commercial Serum Replacement additives are known, such as KonckOut Serum Replacement (KSR), N2, B27, Insulin-Transferrin-sereniumsupplement (ITS), G5, and the like, which are readily available to those skilled in the art. Preferably, the serum-substituting additive used herein is a mixed additive obtained by mixing N2, B27 and/or KSR in a certain proportion, more preferably, the concentration of N2 is between 0% and 1%, the concentration of B27 is between 0% and 2%, and the concentration of KSR is between 0% and 15%; the most preferred embodiment is a concentration of N2 of 1% in the final medium. The above% refers to volume concentration.

The growth factor used in the invention comprises bFGF, Lif, or a mixture of bFGF and Lif; preferably, the concentration of bFGF in the final medium is between 0-10ng/ml and the concentration of Lif is between 500-2000 units/ml; more preferably, the concentration of bFGF in the final medium is 5ng/ml and the concentration of Lif is 2000 units/ml.

The small molecule compound used in the invention is vitamin C. In the medium of the present invention, it is preferable that the concentration of vitamin C added to the medium may be between 10 and 200. mu.g/ml, and more preferably, the concentration of vitamin C is 50. mu.g/ml.

The compounds added in the basic serum-free culture medium comprise ethanolamine, CDP-ethanolamine and phosphatidylethanolamine or a mixture thereof; preferably, the final culture medium contains ethanolamine at a concentration of 0-2mg/ml, CDP-ethanolamine at a concentration of 0-2mg/ml, and phosphatidylethanolamine at a concentration of 0-2mg/ml, but ethanolamine, CDP-ethanolamine, and phosphatidylethanolamine are not zero at the same time. More preferably, the concentration of ethanolamine in the final medium is 1mg/ml, excluding CDP-ethanolamine and phosphatidylethanolamine.

The serum-free basal medium according to the invention is prepared by conventional techniques known to the person skilled in the art, such as the techniques and conditions for the mixed preparation of serum described in Sambrook, Fritsch and Maniatis, molecular cloning protocols, 3 rd edition (2002).

The term "somatic cell" as used in the present invention is a concept relative to "germ cell" and "embryonic stem cell", which is a cell that is no longer pluripotent and generally has a specific function, generated by differentiation of "embryonic stem cell" or continued development of inner cell mass, and is generally obtained from a fetal mouse or adult mouse located after the blastocyst stage (specifically 3.5 days after fertilization in a mouse), and is generally obtained without obtaining germ cells and sources thereof (such as spermatogonial stem cells, genital ridge stem cells, etc.) that may have pluripotency. The somatic cell used herein is preferably derived from a human, rat or mouse, and more preferably, from a mouse. The somatic cells herein may be any type of somatic cell in the body, preferably fibroblasts. The somatic cell may be any one of: fibroblasts, tail tip fibroblasts, bone marrow-derived monocytes, skeletal muscle cells, adipocytes, peripheral blood mononuclear cells, macrophages, hepatocytes, keratinocytes, oral keratinocytes, hair follicle dermal cells, gastric epithelial cells, lung epithelial cells, synovial cells, kidney cells, skin epithelial cells, osteoblasts, neural stem cells, and dermal cells. The somatic cell can express one or more of Sox2, Klf4, Oct4 and c-Myc. The method for allowing the cell to express Sox2, Klf4, Oct4, c-Myc proteins is not particularly limited, and for example, a gene capable of expressing these proteins may be introduced into the cell. In the following specific example of the invention, the somatic cells were fibroblasts expressing Sox2, Klf4, and Oct4, and did not express c-Myc.

The term "somatic reprogramming" as used herein refers to a phenomenon in which a completely differentiated cell loses its original phenotype and is transformed into a pluripotent stem cell, i.e., a transformation from a somatic cell to a pluripotent stem cell.

The term "mesenchymal to epithelial transition" as used herein refers to the phenomenon of the transition of mesenchymal-like cells to epithelial-like cells.

Induction of pluripotent stem cells: the iPS cell, an induced pluripotent stem cell, is a differentiated cell that reverts to the state of a pluripotent stem cell by treatment with a reprogramming factor.

Unless otherwise specified, various reagents mentioned herein are from Invitrogen or Sigma (Sigma).

The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not specify particular techniques or conditions, and are carried out according to techniques or conditions described in literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruke et al, Huang Petang et al) or according to product instructions. The reagents or apparatus used are not indicated by the manufacturer, but are conventional products available commercially, for example from Illumina.

1. Isolation and culture of Mouse Embryonic Fibroblasts (MEF)

Taking a mouse of 13-14 days of gestational age, dislocating cervical vertebra, killing, soaking in 0.5% benzalkonium bromide for 30s, taking out, and fixing on a sterile anatomical plate paved with absorbent paper with ventral surface facing upwards. The abdomen of the pregnant mouse is cleaned by 75% ethanol, so that the abdominal hair is prevented from being adhered to the uterus and other abdominal organs to cause pollution. And (3) dissecting the abdominal skin layer and the muscle layer of the pregnant mouse by using a sterile surgical instrument, and exposing the abdominal cavity of the mouse. The mouse uterus was rapidly removed by removing fat and connective tissue adhered to the uterus with a new sterile surgical instrument. It was placed in a 10cm petri dish pre-filled with PBS containing the double antibody. Mice were washed 3 times in a clean bench with PBS containing double antibody. The envelope was cut open with sterile ophthalmic scissors, the mouse embryos were separated from the extraembryonic tissue, and the embryos were placed in new 10cm petri dishes pre-loaded with double-antibody PBS. Shearing mouse embryo to 1-3mm with elbow eye scissors³Size. Adding appropriate amount of mixed trypsin of 0.25% and 0.05% into the culture dish, digesting for 10min at 37 deg.C until the solution becomes turbid, adding equal volume of MEF cell culture medium, and terminating digestion. Lightly beating and mixing for 5-10 times, and standing. The cell suspension was transferred to a 50ml centrifuge tube, 200g, and centrifuged for 5 min. Discarding supernatant, adding MEF cell culture medium into the precipitate, gently blowing and resuspending, seeding cells into 15cm culture dish, supplementing appropriate amount of culture medium, mixing, and 5% CO at 37 deg.C₂Culturing under the condition. On the next day, the cell status was observed, and the medium was replaced with fresh medium, and the number of labeled cell passages was P0. MEF cell culture medium (500ml) contained: 430ml of high-glucose DMEM medium, 60ml of FBS, 5ml of GlutaMax and 5ml of NEAA respectively.

2. Viral packaging and infection

The cells used for transfection were the Plat-E virus packaging cell line. Plat-E cells were seeded into culture dishes: the 10cm dish is 800 ten thousand; 320 ten thousand micrometer discs; six orifice plates 150 million per orifice. The amount of DNA required corresponds to 10. mu.g; 4 micrograms; 2 microgram.

Cells can be transfected after being inoculated for 12 hours or overnight; cell density was observed to be 70% -90% before transfection. Fresh MEF medium was changed before transfection: a 10cm dish of 7.5 ml; a six centimeter disk of 3 milliliters; six well plates were 1.5 ml per well.

The amounts of the various reagents used at the time of transfection are as follows:

	10cm dish	6 cm dish	Six orifice plates per orifice
				DNA
	10 microgram of	4 microgram of	2 microgram of
				PEI	40 microliter	16 microliter	8 microliter
OPTI-MEM	1 ml of	400 microliter	200 microliter

When in transfection, the DNA and the OPTI-MEM are firstly added and mixed evenly, then the PEI is added and mixed evenly, the mixture is violently reversed and mixed evenly, then the mixture is kept still for 13 to 15 minutes, and then the mixture is added into the Plat-E cells with the changed solution for marking. The dish was gently shaken to homogenize it. The cells were placed in an incubator. After 8-10 hours, cells were replaced with MEF medium: 10cm dish is 10 ml; a six centimeter disk of 4 milliliters; six well plates 2 ml per well.

After Plat-E cells were transfected for 48 hours, the virus was packaged. At this point, the supernatant was collected by syringe and the dish was refilled with the appropriate amount of fresh medium (10 ml in a 10cm dish). Meanwhile, the collected supernatant was filtered through a 0.45 μm filter into a centrifuge tube, and a proper amount of fresh medium (about 12 ml in a 10cm dish total volume) was added, polybrene was added and mixed, and the mixture was stored at 4 ℃ or immediately used as an infection. After another 24 hours, the virus was collected in the same manner as a secondary infection. The cells at this point can be discarded.

The collected virus can infect cells prepared in advance according to the experimental requirements. 6 ml of virus is needed for a 10cm dish; a six centimeter disk of 3 milliliters with a six well plate of 2 milliliters per well.

3. Preparation of a culture medium:

the serum-free culture medium comprises a basal culture medium DMEM and a serum substitute additive N₂(content: 1%), growth factor bFGF (concentration: 5ng/ml) and Lif (concentration: 2000units/ml), small molecule compound vitamin C (concentration: 50. mu.g/ml) and compound ethanolamine (1 mg/ml).

4. Somatic reprogramming

Somatic cell reprogramming is mainly achieved by infecting cells with exogenous viruses carrying Sox2, Klf4 and Oct 4. Generally, the same amount of each virus is used.

The MEF cells after secondary infection are replaced by various induction culture media, and the basic culture medium DMEM is added with serum substitute additive N₂(content: 1%), growth factor bFGF (concentration: 5ng/ml), Lif (concentration: 2000units/ml), and small molecule compound vitamin C (concentration: 50. mu.g/ml) were added as controls, ethanolamine (1mg/ml), CDP-ethanolamine (1mg/ml), phosphatidylethanolamine (1 mg/m/ml), and the likel) as treatment group. Thereafter, fresh medium was changed daily until the desired number of days (8-10 days). If induction efficiency of iPS cells is to be measured, OG2-MEF is used. When the iPSCs clone had GFP fluorescence, it was shown that expression of endogenous Oct4 was a marker for successful reprogramming. At this time, GFP fluorescence positive clones, i.e., the induction efficiency of iPS cells, can be counted under a microscope. Note that a GFP positive clone means that most or all of the cells of the clone emit green fluorescence; meanwhile, when comparison is needed, the judgment standards of GFP positive clones are consistent among different wells, and 3 auxiliary wells are needed in each experiment to averagely reduce errors.

Immunofluorescence, chimeric murine, Western, fluorescent quantitative PCR, and the like are conventional techniques known in the art and will not be described in detail herein.

Example 1: ethanolamine, CDP-ethanolamine, phosphatidylethanolamine can improve mouse fibroblast reprogramming efficiency

As shown in FIG. 1, the culture medium contains DMEM as basic medium and N as serum substitute additive₂Ethanolamine (1mg/ml), CDP-ethanolamine (1mg/ml) and phosphatidylethanolamine (1mg/ml) are respectively added into culture media of growth factors bFGF (concentration of 5ng/ml), Lif (concentration of 2000units/ml) and small molecular compound vitamin C (concentration of 50 mu g/ml), and after 8 days of culture, the reprogramming efficiency of mouse embryo fibroblasts can be improved, and the clone number of induced pluripotent stem cells is greatly increased (figure 1A). The pluripotency of the pluripotent stem cells obtained by reprogramming can be identified by utilizing immunofluorescence and chimeric mouse methods, and the result of immunofluorescence experiments shows that the induced pluripotent stem cells obtained from the control group and the ethanolamine group both express pluripotency proteins Oct4, Rex1 and SSEA1 (figure 1B). The chimeric mouse experiment shows that the induced pluripotent stem cells obtained from the control group and the ethanolamine group can generate the chimeric mouse with reproductive transmission capacity, # mark is the chimeric mouse and # mark is the progeny of the chimeric mouse (FIG. 1C).

Example 2: ethanolamine can improve the reprogramming efficiency of mouse tail fibroblast cells and can improve the reprogramming efficiency in a basic serum-free culture medium containing KSR.

As shown in FIG. 2, in the medium containing the basal cultureBasal DMEM, serum replacement additive N₂Ethanolamine (1mg/ml) is added into a culture medium containing (the content is 1 percent), growth factors bFGF (the concentration is 5ng/ml), Lif (the concentration is 2000units/ml) and a small molecular compound vitamin C (the concentration is 50 mu g/ml), and after the mouse tail tip fibroblasts are cultured for 10 days, the reprogramming efficiency of the mouse tail tip fibroblasts can be improved, so that the clone number of induced pluripotent stem cells is greatly increased (figure 2A). Ethanolamine (1mg/ml) is added into a culture medium containing a basal culture medium DMEM, a serum replacement additive KSR (content is 15%), a growth factor bFGF (concentration is 5ng/ml), Lif (concentration is 2000units/ml) and a small molecular compound vitamin C (concentration is 50 mu g/ml), and after the mouse tail tip fibroblasts are cultured for 10 days, the reprogramming efficiency of the mouse tail tip fibroblasts can be improved, so that the clone number of induced pluripotent stem cells is greatly increased (figure 2B).

Example 3: ethanolamine can promote the mesenchymal epithelial transition of mouse embryonic fibroblasts.

As shown in FIG. 3, the culture medium contains DMEM as a basic medium and N as a serum replacement additive₂Ethanolamine (1mg/ml) is added into a culture medium containing (the content is 1 percent), growth factors bFGF (the concentration is 5ng/ml), Lif (the concentration is 2000units/ml) and a small molecular compound vitamin C (the concentration is 50 mu g/ml), and after the mouse embryonic fibroblasts are cultured for 2 days, interstitial epithelial transformation of the mouse embryonic fibroblasts can be promoted, the fluorescent quantitative PCR technology is utilized to discover that the ethanolamine promotes the expression of epithelial cell marker genes, and the expression of the interstitial cell marker genes is reduced (figure 3A). The expression of epithelial cell marker proteins of mouse embryonic fibroblasts can be improved by using the Western technology (figure 3B).

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.

Claims

1. A method of reprogramming a somatic cell, comprising the steps of:

preparation of a Medium for Programming: adding a serum substitute additive, a growth factor and a small molecular compound into a serum-free basic culture medium to obtain a serum-free basic culture medium; then adding the compound into the serum-free basal medium, and reversing and mixing uniformly; obtaining a prepared culture medium for programming; the serum-substituting additive is N₂At least one of B27, KSR, said growth factor comprising at least one of bFGF and Lif, said small molecule compound comprising vitamin C; the compound is at least one of ethanolamine, CDP-ethanolamine and phosphatidylethanolamine;

and culturing the somatic cells in the programming culture medium, wherein the cells can generate mesenchymal transition to epithelial transition after about 2 days, and the induced pluripotent stem cells are obtained after about 8-10 days.

2. The method of reprogramming a somatic cell of claim 1, wherein N is₂The concentration of the KSR is 0-1%, the concentration of the B27 is 0-2%, the concentration of the KSR is 0-15%, and the three are not 0 at the same time.

3. The method for reprogramming somatic cells according to claim 1, wherein the concentration of bFGF is 0-10ng/ml, and the concentration of Lif is 500-2000 units/ml; more preferably, bFGF and Lif are included, wherein the concentration of the bFGF is 5ng/ml, and the concentration of the Lif is 2000 units/ml.

4. Method for somatic cell reprogramming according to claim 1, characterized in that the concentration of vitamin C is 10-200 μ g/ml, more preferably the concentration of vitamin C is 50 μ g/ml.

5. The method of reprogramming somatic cells of any one of claims 1-4, wherein the concentration of ethanolamine is 0-2mg/ml, the concentration of CDP-ethanolamine is 0-2mg/ml, the concentration of phosphatidylethanolamine is 0-2mg/ml, and the three are not 0 at the same time.

6. The method of somatic cell reprogramming of claim 5, wherein the concentration of said ethanolamine is 1mg/ml, the concentration of said CDP-ethanolamine is 1mg/ml, the concentration of said phosphatidylethanolamine is 1 mg/ml; preferably, the serum replacement additive is N at a concentration of 1%₂。

7. The method for reprogramming somatic cells according to claim 5, wherein the basal medium is one of DMEM medium, MEM medium and α MEM medium, preferably DMEM.

8. The method of reprogramming somatic cells of claim 5, wherein the somatic cells express Sox2, Klf4, and Oct4, and do not express c-Myc.

9. Method for somatic cell reprogramming according to claim 5, characterized in that the somatic cell is any type of somatic cell in the body, preferably a fibroblast.

10. Induced pluripotent stem cells prepared by the method for reprogramming somatic cells according to claims 1 to 9.