CN105505859B

CN105505859B - Method for inducing and differentiating fibroblast into multifunctional stem cell

Info

Publication number: CN105505859B
Application number: CN201510987834.0A
Authority: CN
Inventors: 李遇梅; 张怡萱; 李淑; 刘莉萍; 许辉; 惠利键; 郑允文
Original assignee: Affiliated Hospital of Jiangsu University
Current assignee: Affiliated Hospital of Jiangsu University
Priority date: 2015-12-28
Filing date: 2015-12-28
Publication date: 2020-02-21
Anticipated expiration: 2035-12-28
Also published as: CN105505859A

Abstract

The invention relates to a method for inducing fibroblasts into multifunctional stem cells, in particular to a method for inducing the fibroblasts at skin lesions of patients with leucoderma into the multifunctional stem cells, which belongs to the technical field of biology; the invention adopts a four-factor introduction method to induce the fibroblasts at the skin lesion of the leucoderma patient into multifunctional stem cells, identifies the induced cells, and determines that the cells have the characteristics of the stem cells and are similar to ES cells; the source of the ES cells is in ethical dispute, the multifunctional stem cells induced by the patient body cells are wide in source, have no immunological rejection, have corresponding disease characteristics, and can be used for disease research, drug screening and the like; the invention can induce multifunctional stem cells from somatic cells of various patients, and the invention uses the fibroblasts at the skin lesion of the leucoderma patients for the first time to induce the multifunctional stem cells.

Description

Method for inducing and differentiating fibroblast into multifunctional stem cell

Technical Field

The invention relates to a method for inducing and differentiating fibroblasts into multifunctional stem cells, in particular to a method for inducing skin fibroblasts at skin lesions of patients with leucoderma into multifunctional stem cells, and belongs to the technical field of biology.

Background

Vitiligo is a common local or general depigmentation skin disease, the incidence rate of the disease is about 0.5-2%, and clinically, the disease is manifested by depigmentation and leukoplakia, and the edge is clearer. The pathogenesis of vitiligo is not well defined, and in recent years, research is related to genetic factors, autoimmune factors, oxidative stress, melanocyte self-destruction, psychoneural factors and the like. The treatment method of the disease has various methods including drug treatment, physical treatment, surgical treatment and the like, but the effect is not ideal and the life of the patient is influenced to a certain extent.

Research on treatment of diseases with stem cells has been carried out with many efforts in recent years, and treatment of vitiligo can also be achieved with stem cells. Because the function of the melanocyte at the skin lesion of the leucoderma patient is reduced or even lost, the melanocyte can be induced by using stem cells for relevant treatment. The stem cells inducing and differentiating the melanocytes at present include embryonic stem cells (EScell), mues cells (Muse cell), Mesenchymal Stem Cells (MSC) and induced pluripotent stem cells (iPS cell).

Induction of pluripotent stem cells was first induced by Yamanaka, a scientist of japan, using mouse fibroblasts (Takahashi, k. and s. Yamanaka,Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.cell, 2006.126(4): p.663-76), cells are changed from a terminal differentiation state to a stem Cell state by over-expressing four transcription factors of Oct4, Sox2, Klf4, c-Myc. Later, induction into stem cells was successfully achieved using human fibroblasts. The iPS cell is similar to the ES cell in aspects of morphology, epigenetics, function and the like, but avoids the ethical problems of the ES cell.

Since the advent of iPS technology, it has become a global research hotspot. Many laboratories around the world gradually use somatic cells from different patient sources to reprogram corresponding iPS cells, and the cells not only have characteristic changes of corresponding diseases, but also have important significance in the aspects of establishment of disease models, research, drug screening and the like. Diseases for which the iPS Cell line has been successfully established include gaucher's Disease, parkinson's Disease, trisomy 21 (Park, i.h., et al., Disease-Specific Induced complex step cells, 2008.134 (5): p.877-86.), dilated cardiomyopathy (Sun, n., et al., Patient-Specific Induced complex step cells as a model for a facial differentiated heart Disease, Sci trans Med, 2012.4 (130): p.130 ra 47.), retinitis pigmentosa (Jin, z.b., et al., Modeling recovery Using tissue-Specific tissue step cells, 2011.6 (2), neuroblastoma, leukemia, Cell-tissue, and leukemia (Cell) 2. infiltration, leukemia, Cell 2. infiltration, leukemia, Cell 2. and leukemia (m. host. m., Cell et al.), [ Induced multiplexed step cells from a sheet of patients for differentiating between patients ] Rinsho Ketsueki, 2014.55 (10): p.2202-7.), rheumatoid arthritis (Lee, J.et., et al, Generation of disease-specific Induced multiplexed step cells from a sheet of patients with a rhematoid arthritis and osteo arthritis.Arthrosis Research & Therapy, 2014.16 (1).) and the like, there are fewer examples of reprogramming of patient iPS cells with patient somatic cells in a skin-related disease.

As iPS cells can be derived from various adult cells, the iPS cells of patients become a research hotspot. iPS cells induced by the adult cells of the patients have the characteristics of the diseases, so the iPS cells of the patients have important significance in the aspects of disease treatment, disease model establishment, drug screening and the like. The iPS cells of the patient do not have disease characteristics by means of gene modification and the like, so that the relevant cells, tissues or organs are induced and differentiated to carry out autologous transplantation treatment on the patient, and immune rejection reaction does not occur, which is another advantage of the iPS cells compared with ES cells. At present, the body cells of various patients are induced into multifunctional cells, and the induction of the body cells of patients suffering from leucoderma into the multifunctional stem cells is not reported in documents.

Disclosure of Invention

The invention aims to provide a method for inducing fibroblasts at skin lesions of patients with leucoderma into multifunctional stem cells.

The invention provides a method for inducing fibroblasts at skin lesion of a leucoderma patient into multifunctional stem cells for the first time, which is characterized by comprising the following steps: obtaining fibroblasts at skin lesion of a leucoderma patient; induction of multifunctional stem cells of leucoderma patients; and identifying the obtained leucoderma patient induced multifunctional stem cells.

(1) Obtaining fibroblasts at skin lesion of a leucoderma patient:

A. after the skin lesion tissue of the leucoderma patient is obtained, the tissue is taken out from a super clean bench and is treated with a solution containing 1 percent of double antibiotics (penicillin and streptomycin)

Mycin) washing with PBS buffer solution for 3 times, and cutting into pieces of 1mm³And (5) small blocks.

B. Covering the tissue block with sterilized cover glass, and adding high-glucose DMEM medium containing 10% serum

(Hyclone) was cultured at 37 degrees, fibroblasts appeared after 10 to 15 days, and the culture medium was changed after 2 to 3 days.

(2) Induction of multifunctional stem cells of leucoderma patients:

A. taking 10 fibroblasts from the skin lesion of the leucoderma patient obtained in the step (1)⁵Adding Oct4,

Four retroviruses of Sox2, Klf4 and c-Myc are infected, the virus amount is 400 mu L respectively, and four transcription factors of Oct4, Sox2, Klf4 and c-Myc are overexpressed.

B. Transferring the cultured cells to feeder layer cells (mouse embryo fibroblasts) after 24h, and replacing the cells

Introducing culture medium at 37 deg.C and 5% CO₂、5%O₂The cells were photographed by changing the medium every day and observing the cell morphology under a microscope, and after 12 days, the cells were monocloned onto new feeder cells (mouse embryonic fibroblasts).

Wherein the inducing medium comprises ES medium added with vitamin C (50 mg/ml), and ES medium bag

Comprises 80% DMEM/F12, 20% KSR, 1% Non-essential amino acids, 1mM L-glutaMAX, 0.1mM β -mercaptoethanol and 5ng/ml bFGF.

C. Culturing with ES medium in a low-oxygen incubator at 37 deg.C and 5% CO₂、5%O₂，

The medium was changed every day, and the cell morphology was observed microscopically and photographed.

(3) Identifying the characteristics of the leucoderma patients for inducing the multifunctional stem cells and determining the characteristics of the stem cells to

And a function.

Technical advantages of the invention

(1) iPS cells are induced multifunctional stem cells similar to ES cells and are used in epigenetic inheritance,

The ES cells have high similarity in functions and the like, but the ES cells are controversial since the advent because human ES cells are derived from aborted fetuses, are not ethical and are not suitable for mass production, and create obstacles to their application, but the iPS cells do not have such problems in terms of sources, and iPS cell lines have been established by various cells, including fibroblasts, lymphocytes, liver cells, keratinocytes, even urine cells and the like;

(2) the iPS cells not only have wide sources, but also can avoid immunological rejection reaction in the process of utilizing the iPS cells

The human ES cells are also allogeneic cells, the ES cells have the problem of immunological rejection when used for treating diseases, and the autologous iPS cells of the patient cannot generate immunological rejection reaction in the treatment process.

(3) iPS cells from the patient demonstrated the characteristics of the corresponding disease by the administration of iPS to the patient

The study can clearly understand the defects of the disease in certain gene, and is beneficial to the study of the disease. Meanwhile, a corresponding disease model can be established by using the patient iPS cell and is input into a mouse body to establish a humanized mouse disease model, and the research on aspects such as drug screening and the like can be carried out.

(4) The establishment of the patient iPS cell line becomes a hot spot, and a plurality of diseases are established in a plurality of laboratories

Such as gaucher's disease, parkinson's disease, trisomy 21 syndrome, dilated cardiomyopathy, retinitis pigmentosa, neurofibromatosis, leukemia, rheumatoid arthritis, and the like. In dermatological studies, it is also possible to use this technique to study related diseases, but there are currently few examples and the study of diseases in terms of depigmentation is more rare. The invention firstly utilizes the fibroblasts at the skin lesion of the leucoderma patients to induce the multifunctional stem cells, and has important significance for the stem cell treatment of the leucoderma and the research on the pathogenesis of the leucoderma.

Drawings

FIG. 1 is a diagram of the morphology of fibroblasts at the skin lesion of a patient with vitiligo, wherein the scale size is 100 μm;

FIG. 2, observation of cell morphology ('4') of day 4 during the process of inducing fibroblasts into multifunctional stem cells at skin lesions of vitiligo patients, wherein the arrows indicate the occurring small clones of iPS; the sizes of the scales in the 2 figures are all 100 micrometers;

FIG. 3 is a diagram of the morphological observation of iPS (VP-iPS) cells of a second generation vitiligo patient; a: x 4; b: x 10; the scale sizes in the graphs a and b are both 100 μm;

FIG. 4 shows the result of alkaline phosphatase staining of VP-iPS cells; a: x 4; b: x 10; the scale size in panels a and b is 100 μm;

FIG. 5 shows the expression result of the VP-iPS cell immunofluorescence staining detection stem cell surface marker; markers include OCT4, NANOG, TRA-1-60, SSEA 4;

FIG. 6 is a relative expression pattern of the VP-iPS cells in the stem cell-associated genes (P < 0.05);

FIG. 7 VP-iPS cells are in demethylated state in the OCT4 promoter region;

FIG. 8 shows the result of karyotype analysis of VP-iPS cells.

Detailed Description

The invention is further illustrated by the following examples.

The reagent materials required are the names of the source companies in parentheses;

DMEM/F12 (Gibco), KOSR (Gibco), Non-essential amino acids (Gibco), L-glutamax (Gibco), β -mercapthanol (Gibco), bFGF (Peprotech), DMEM (Hyclone), FBS (Gibco), Vc (Sigma), polynene (Sigma), GCDR (Stemcell), PBS (Hyclone), Y27632 (Selleck), AP reagent (Sigma), goat anti-human OCT4 (Santa Cruz), rabbit anti-human NANOG (Gentex), mouse anti-human TRA-1-60 (MilliMilpore), SSEA4 (dshb), donkey anti-sheep CY3, donkey anti-rabbit CY3, donkey anti-mouse CY5 (Jacpsson), genome Bilite (Kifir), and reverse transcription Kit (Turbo-PCR).

ES medium comprises 80% DMEM/F12, 20% KSR, 1% Non-essential amino acids, 1mM L-glutaMAX, 0.1mM β -mercaptoethanol, 5ng/mL bFGF.

The method for inducing the fibroblasts at the skin lesion of the leucoderma patient into the multifunctional stem cells comprises the following steps:

(1) obtaining fibroblast at skin lesion of leucoderma patient

A. After the skin lesion tissue of the leucoderma patient is obtained, the tissue is taken out from a super clean bench and is treated with a solution containing 1 percent of double antibiotics (penicillin and penicillin)

Streptomycin) PBS buffer solution for 3 times, and cutting into 1mm pieces³And (5) small blocks.

The morphology of the fibroblasts in the skin lesion of the vitiligo patient is observed to be long spindle, as shown in figure 1.

(2) The method comprises the following steps of inducing fibroblasts at the skin lesion of a leucoderma patient into multifunctional stem cells:

A. taking 10% of fibroblasts from skin lesion of leucoderma patient⁵Adding Oct4, Sox2, Klf4, and,

c-Myc, wherein the virus amount is 400 mu L, and four transcription factors of Oct4, Sox2, Klf4 and c-Myc are overexpressed.

B. Transferring the cultured cells to feeder layer cells (mouse embryo fibroblasts) after 24h, and replacing and inducing

Culture medium at 37 deg.C and 5% CO₂、5%O₂The cells were photographed by changing the medium every day and observing the cell morphology under a microscope, and after 12 days, the cells were monocloned onto new feeder cells (mouse embryonic fibroblasts).

Wherein the inducing medium comprises ES medium containing vitamin C (50 mg/mL) and ES medium

80% DMEM/F12、20% KSR、1% Non-essential amino acids、1mM L-glutaMAX、0.1mM β-mercaptoethanol、5ng/ml bFGF。

C, VP-iPS cell culture: cultured in ES medium comprising 80% DMEM/F12, 20% KSR,

1% Non-essential amino acids, 1mM L-glutaMAX, 0.1mM β -mercaptoethanol, 5ng/ml bFGF were placed in a hypoxic incubator at 37 ℃ with 5% CO₂、5%O₂The medium was changed every day, and the cell morphology was observed microscopically and photographed.

The iPS cell clone morphology appears at the 4 th day of the induction period, as shown in figure 2, iPS cell transmission of leucoderma patients

Cells appeared in a compact, clone-like morphology after the second generation, as shown in FIG. 3.

(3) The induced leucoderma patient iPS cell (VP-iPS cell) is identified to have stem cell characteristics in the following aspects:

A. alkaline phosphatase staining

Among ES cells, undifferentiated cells were stained purple red, and the staining result of VP-iPS cells was purple red according to the procedure of alkaline phosphatase staining kit (Sigma). The results are shown in fig. 4, and are positive results, indicating that iPS cells are undifferentiated, similar to ES cells.

B. Immunofluorescence staining

The stem cell surface markers OCT4, TRA-1-60, NANOG and SSEA4 are detected in VP-iPS cells by the following steps: after cells were fixed in 4% PFA for 15 minutes, they were permeabilized with 0.25% TritonX-100, washed 3 times with PBS, 3% BSA was added for blocking for one hour, and then primary anti-goat anti-human OCT4 (Santa Cruz Co.), rabbit anti-human NANOG (Genetex Co.), mouse anti-human TRA-1-60 (Millipore Co.), SSEA4 (dshb Co.), incubated overnight at 4 ℃ after sucking out the primary antibody, washed three times with PBST, added with secondary anti-donkey anti-goat CY3, donkey anti-rabbit CY3, donkey anti-mouse CY5 (Jackson Co.), incubated in the dark for one hour, and the results were observed under an inverted fluorescence microscope. The results showed that OCT4 and NANOG fluoresced red, TRA-1-60 and SSEA4 fluoresced green, and 4 surface markers were positively expressed, as shown in FIG. 5.

C. Fluorescent quantitative PCR detection of stem cell related gene expression

After passage of the VP-iPS cells to the 6 th generation, fluorescent quantitative PCR detection is carried out, mRNA expression levels of stem cell related genes OCT4, SOX2, NANOG and REX1 are compared, the expression level of the VP-iPS cells is similar to that of the ES cells by taking the expression level of the ES cells as a positive reference, and the expression level of the VP-iPS cells is remarkably improved (P is less than 0.05) for a negative reference leucoderma patient fibroblast (VPDF) group, and the result is shown in FIG. 6.

Detection of OCT4 promoter region demethylation

And carrying out demethylation detection on the OCT4 promoter region after the VP-iPS cell is passaged to the 11 th generation, and connecting the cell extracted DNA to a PMD18 vector for sequencing after bisulfite treatment. In FIG. 7, a white circle indicates a demethylation state, a black circle indicates a methylation state, and the result analysis results show that the VP-iPS gene is entirely demethylated in the OCT4 gene promoter region for the negative reference leucoderma patient fibroblast (VPDF) group.

E. Karyotyping analysis

With the generation of iPS cells, mutation and other problems can occur, the 11 th generation VP-iPS cells are sent to a related karyotype identification department for chromosome karyotype analysis, and the stable and normal karyotype of the iPS cells of the leucoderma patients is determined, and the result is shown in figure 8.

Claims

1. A method for inducing fibroblasts into pluripotent stem cells is characterized in that the pluripotent stem cells are derived from fibroblasts at skin lesions of patients with leucoderma, and the specific method comprises the following steps:

(1) obtaining fibroblasts at skin lesion of a leucoderma patient;

(2) the induction of the multifunctional stem cells of the leucoderma patients comprises the following specific steps:

A. adding retrovirus Oct4, Sox2, Klf4 and c-Myc into fibroblasts at the skin lesion of the vitiligo patient obtained in the step (1) to infect, and overexpressing the retrovirus;

the addition amount of the four retroviruses is 10 percent of the fibroblasts at the skin lesion of the leucoderma patient⁵Adding 400 mu L of retrovirus into the cell amount;

B. transferring the cells to feeder layer cells after 24h of culture, replacing an induction culture medium for culture, changing liquid every day, observing cell morphology by a microscope for photographing, and picking monoclonals to new feeder layer cells after 12 days;

the induction culture medium is an ES culture medium and vitamin C, wherein the ES culture medium comprises 80% DMEM/F12, 20% KSR, 1% Non-essential amino acids, 1mM L-glutaMAX, 0.1mM β -mercaptoethanol and 5ng/mLbFGF, and the content of the vitamin C is 50 mg/mL;

C. culturing with an induction culture medium, placing in a hypoxia culture box, changing the culture medium every day, observing the cell morphology with a microscope and taking a picture;

(3) and identifying the obtained leucoderma patient induced multifunctional stem cells.

2. The method of claim 1, wherein the fibroblast cells are induced into pluripotent stem cells,

the method for obtaining the fibroblasts at the skin lesion of the vitiligo patient in the step (1) comprises the following specific steps:

A. after skin lesion tissues of the vitiligo patients are obtained, the vitiligo patients are taken out from an ultra-clean bench, washed by PBS (phosphate buffered saline) containing 1% double antibody and then cut into small pieces;

B. and covering the tissue block with a sterilized cover glass, adding a culture medium for culturing, wherein fibroblasts appear after 10 to 15 days, and changing the culture solution after 2 to 3 days.

3. The method of claim 1, wherein the fibroblast cells are induced into pluripotent stem cells,

the culture medium in the step B is a high-sugar DMEM culture medium containing 10% serum.

4. The method of claim 1, wherein the feeder cells in step B are mouse embryonic fibroblasts.