WO2017121386A1 - Method for preparing pluripotent stem cells of exogenous small-rna and usage thereof - Google Patents

Abstract

Provided is a method for preparing pluripotent stem cells of exogenous small -RNA. Also provided is a usage of exogenous small-RNA for preparing a reagent kit for facilitating conversion of somatic cells or specialized stem cells into multipotent stem cells.

Description

Method for preparing pluripotent stem cells by exogenous small RNA and use thereof Technical field

The present invention relates to the field of molecular biology, and in particular to a method and use of exogenous small RNA for preparing pluripotent stem cells.

Background technique

The pluripotent stem cell has the potential to differentiate into a variety of cellular tissues, which can differentiate into all cells in the body, thereby forming all tissues and organs of the body. Therefore, the study of pluripotent stem cells not only has important theoretical significance, but also has great application value in organ regeneration, repair and disease treatment.

In the past, people have always believed that pluripotent stem cells can only be obtained from human embryos. However, in 2007, scientists in the United States and Japan found that the application of human and mouse normal skin cells, the introduction of KLF4, OCT4, SOX2 and C-MYC four genes, can be converted from normal somatic cells into pluripotent stem cells. The pluripotent stem cells induced by this gene are called Induced Pluripotent Stem Cells (iPs). In addition to skin cells, other somatic cells can also produce iPs. iPs have been successfully cultured and differentiated into various somatic cells and different tissues such as myocardium, nerves, pancreas and bone. iPS technology is a major breakthrough in the field of stem cell research. It avoids the long-standing ethical controversy, solves the problem of immune rejection in stem cell transplantation medicine, and makes stem cells a big step in clinical application. However, in the past, induced pluripotent stem cells must use retroviral vectors for genome integration. Due to the random nature of genomic integration, mutations can occur and can even cause cancer and genetic diseases. Finding an efficient way to promote the transformation of somatic cells into pluripotent stem cells is an urgent problem to be solved.

Pluripotent stem cells have also become a hot spot and focus of current stem cell research. Some reports on the induction of iPs, using small molecule combination treatment to contact differentiated cells with TGF-beta receptor inhibitors, GSK3-beta inhibitors and cAMP activators, whereby the differentiated cells undergo cell fate reprogramming, resulting in Induced pluripotent stem cells acquire fully reprogrammed iPSCs, but the method is poorly reproducible. Japanese scientists exposed cells isolated from newborn mice to a weakly acidic environment to restore the cells to an undifferentiated state and have the potential to differentiate into any cell type, but this pure physical stimulation enables differentiated cells. The discovery of the phenomenon of returning to the pluripotent state has been greatly questioned.

Therefore, there is an urgent need for a method for efficiently and reproducibly preparing pluripotent stem cells.

Summary of the invention

It is an object of the present invention to provide a method for efficiently and reproducibly preparing pluripotent stem cells.

A first aspect of the invention provides the use of an exogenous small RNA molecule for the preparation of a reagent or kit for promoting the conversion of somatic or multipotent stem cells into pluripotent stem cells.

In another preferred embodiment, the reagent comprises a transfection reagent.

In another preferred embodiment, the somatic cells include: epithelial cells, nerve cells, red blood cells, white fine Cells, platelets, phagocytic cells (phagocytic neutrophils, basophils, phagocytic granulocytes, etc.), B lymphocytes, effector B cells, memory B cells, T lymphocytes, memory T cells, effector T cells, Cardiomyocytes, smooth muscle cells, skeletal muscle cells, cardiomyocytes, osteoblasts, glial cells, hepatocytes, kidney cells, gland cells, endocrine cells (thyroid cells, thymocytes, islet B cells, islet cells, etc.).

In another preferred embodiment, the pluripotent stem cells include: hematopoietic stem cells, embryonic stem cells, bone marrow mesenchymal stem cells, neural stem cells, muscle stem cells, osteogenic stem cells, endoderm stem cells, retinal stem cells, pancreatic stem cells.

In another preferred embodiment, the small RNA molecule is selected from the group consisting of miRNA, siRNA- or a combination thereof.

In another preferred embodiment, the small RNA molecule comprises at least two or all of the miRNAs selected from the group consisting of (A): miR-432, miR-320, miR-27b, miR-103.

In another preferred embodiment, the small RNA molecule further comprises at least one or all of the miRNAs selected from the group consisting of: miR-423, miR-185, miR-378, miR-130b.

In another preferred embodiment, the small RNA molecule further comprises at least one or all of the miRNAs selected from the group (C): let-7g, miR-107.

In another preferred embodiment, the small RNA molecule comprises ≥4 species, preferably ≥6 species, more preferably ≥7 species, selected from the group (A), the group (B), and/or the group (C). Optimally ≥ 8 or all miRNAs.

In another preferred embodiment, the small RNA molecule comprises at least three or all of the miRNAs selected from the group (A). .

In another preferred embodiment, the small RNA molecule comprises at least four or all of the miRNAs selected from the group consisting of: hsa-miR-432-5p, hsa-miR-320a, hsa-miR-27b-3p, hsa-miR-103a-3p, hsa-miR-423-5p, hsa-miR-185-5p, hsa-miR-378a-3p, hsa-let-7g-5p, hsa-miR-130b-3p, or combination.

In another preferred embodiment, the small RNA molecule comprises at least 4 or all of the miRNAs selected from the group consisting of: bta-miR-107, bta-miR-432, bta-miR-320a, bta-miR- 27b, bta-miR-103, bta-miR-423-3p, bta-miR-423-5p, bta-miR-185, bta-miR-378, bta-let-7g, bta-miR-130b, or combination.

In another preferred embodiment, the small RNA molecule comprises at least 4 or all of the miRNAs selected from the group consisting of: mmu-miR-320-3p, mmu-miR-27b-3p, mmu-miR-103- 3p, mmu-miR-423-5p, mmu-miR-185-5p, mmu-let-7g-5p, mmu-miR-130b-3p, or a combination thereof.

In another preferred embodiment, the small RNA molecule comprises at least one or all of the miRNAs selected from the group consisting of: miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369- 3p, miR-369-5p, miR-21, miR-214, or a combination thereof.

In another preferred embodiment, the agent is also used to upregulate expression of a transcription factor in the cell.

In another preferred embodiment, the transcription factor is selected from the group consisting of Oct4, Sox2, c-Myc, n-Myc, Klf4, Nanog, Lin28, Zeb2, Mecp2, p21, wisp1, mbd2, p53, Jun, or combination.

In another preferred embodiment, the transcription factor is selected from the group consisting of Oct4, Sox2, c-Myc, n-Myc, Klf4, Nanog, Lin28, or a combination thereof.

In another preferred embodiment, the transcription factor is selected from the group consisting of Oct4, Sox2, c-Myc, Klf4, or a combination thereof.

In another preferred embodiment, the transcription factor is selected from the group consisting of Zeb2, Mecp2, p21, wisp1, mbd2, p53, Jun, or a combination thereof.

In another preferred embodiment, the transcription factor is selected from the group consisting of mbd2, p53, Jun, or a combination thereof.

In another preferred embodiment, the small RNA molecule is synthetic, recombinant, or naturally occurring.

In another preferred embodiment, the small RNA molecule is derived from humans, cows, pigs, sheep, rodents.

In another preferred embodiment, the small RNA molecule has a size of from 12 to 80 nt, preferably from 14 to 60 nt, more preferably from 15 to 30 nt, most preferably from 18 to 22 nt.

In another preferred embodiment, the mammal comprises a human or a non-human mammal.

In another preferred embodiment, the non-human mammal comprises a rodent such as a mouse or a rat.

In another preferred embodiment, the transfection reagent comprises or is a pH acidic regulator.

In another preferred embodiment, the transfection reagent comprises or is a microparticle preparation.

In another preferred embodiment, the microparticle formulation comprises microparticles and the small RNA molecule encapsulated in the microparticles.

A second aspect of the invention provides a method for promoting the transformation of somatic or multipotent stem cells into pluripotent stem cells in vitro, comprising the steps of:

(i) providing a transfection system comprising: (a) a buffer and/or a culture; (b) somatic or multipotent stem cells; and (c) small RNA;

(ii) placing the transfection system under transfection conditions such that the small molecule RNA is transfected into the somatic or multipotent stem cells;

(iii) inducing culture of the transfected somatic cells or multipotent stem cells obtained in the previous step, thereby converting the somatic cells or the multipotent stem cells into pluripotent stem cells.

In another preferred embodiment, the transfection system is an aqueous system.

In another preferred embodiment, the cell is a mammalian cell.

In another preferred embodiment, in step (ii), the transfection conditions comprise an acidic pH treatment.

In another preferred embodiment, the acidic pH treatment refers to treatment at a pH of 2.5 to 6.0, preferably 2.8 to 5.5, more preferably 2.9 to 4.5, most preferably 3.0 to 4.0.

In another preferred embodiment, the acidic pH treatment is carried out for a period of from 0.5 minutes to 24 hours.

In another preferred embodiment, the treatment time of step (ii) is from 0.5 minutes to 24 hours, preferably from 1 minute to 6 hours, more preferably from 5 minutes to 2 hours, most preferably from 0.25 to 1 hour.

In another preferred embodiment, in the step (ii) and/or (iii), the temperature is 4 to 50 ° C, preferably 15 to 45 ° C, more preferably 25 to 40 ° C, most preferably 35 to 39 ° C. .

In another preferred embodiment, in step (iii), comprising: transfecting the somatic cells obtained in the previous step Or a competent stem cell is transferred into a somatic cell of the exogenous nucleic acid, and induced to obtain a pluripotent stem cell.

In another preferred embodiment, in step (iii), the induced culture comprises the following conditions: plasmid vector mediated, calcium phosphate coprecipitation, electroporation, DEAE-dextran and polybrene, mechanical method ( Microinjection and gene gun), cationic liposome reagent transfection.

In another preferred embodiment, the method is non-therapeutic and non-diagnostic.

A third aspect of the invention provides a composition or combination for promoting the transformation of somatic or multipotent stem cells into pluripotent stem cells, comprising:

(i) an exogenous small RNA molecule; and

(ii) a pH acidity regulator for providing an acidic pH having a pH of from 2.5 to 6.0.

In another preferred embodiment, the small RNA molecule comprises at least two or all of the miRNAs selected from the group consisting of (A): miR-432, miR-320, miR-27b, miR-103.

A fourth aspect of the invention provides the use of a composition according to the third aspect of the invention for the preparation of a kit for promoting the transformation of somatic cells and/or multipotent stem cells into pluripotent stem cells.

In another preferred embodiment, the kit is further for regulating pluripotent stem cell transcription factor expression.

A fifth aspect of the invention provides a kit, the kit comprising:

(i) a container, and exogenous small RNA molecules located within the container;

(ii) a label or instructions indicating that the kit is for promoting the conversion of somatic cells and/or multipotent stem cells to pluripotent stem cells.

In another preferred embodiment, the kit further comprises (iii) a pH acidity regulator for providing an acidic pH having a pH of from 2.5 to 6.0.

In another preferred embodiment, the pH adjusting agent is an acid.

In another preferred embodiment, the small RNA molecule comprises from 3 to 100, preferably from 4 to 50, more preferably from 5 to 20.

In another preferred embodiment, the small RNA molecules are placed separately or in combination.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

Figure 1 shows the relative content of osa-156a in wild type 293T cells treated with different pH.

Figure 2 shows a schematic view of Lipofectamine ^TM 2000 and acid-induced fluorescent FAM siRNA transfection results.

detailed description

After extensive and intensive research, the inventors discovered for the first time an efficient preparation of pluripotent Cell method. Experiments have shown that by adjusting the eukaryotic cell culture environment to be acidic, small RNA can be more efficiently entered into eukaryotic cells. Moreover, under the action of acid, small RNA can regulate a variety of pluripotent stem cell transcription factors and promote the transformation of somatic cells and/or multipotent stem cells into pluripotent stem cells. The method of the invention is convenient and efficient to operate, does not need to add an exogenous vector, has low toxicity to cells, high transfection rate, and the introduced nucleic acid molecule is more stable. On the basis of this, the present invention has been completed.

Small RNA (small RNA)

In the present invention, the term "small ribonucleic acid (small RNA)" refers to a small fragment of RNA of a length of twenty-five nucleotides; according to the widely accepted classification method proposed by Steven Buckingham in May 2003, small RNA (small RNAs) are non-coding RNAs other than transcribed RNA (including ribosomal RNA and transfer RNA), including microRNAs, short interfering RNAs (siRNA), small nucleolar RNA ( snoRNA) and small nuclear RNA (snRNA).

Among them, microRNAs (miRNAs) are a class of single-stranded small RNA molecules of about 19-23 nucleotides in length, located in the non-coding region of the genome, which are highly conserved in evolution and can inhibit the translation process of target genes. Gene expression is regulated and closely related to many normal physiological activities of animals, such as biological individual development, tissue differentiation, apoptosis and energy metabolism, and is also closely related to the occurrence and development of many diseases. Existing studies have also confirmed that plant miRNAs can also enter the animal through food intake and participate in regulatory activities.

Small interfering RNA (siRNA) is a kind of double-stranded RNA molecule composed of more than 20 nucleotides, which can silence gene expression by messenger RNA (mRNA) which specifically degrades target genes. The role. This process is called RNA interference (RNAi). RNA interference is a way of post-transcriptional regulation of genes. siRNA can specifically recognize its target gene and recruit a protein complex called RNA induced silencing complex (RISC). RISC contains ribonuclease and the like, and can specifically and efficiently inhibit gene expression by targeting cleavage of homologous mRNA. Since RNA interference technology can specifically eliminate or turn off the expression of specific genes, this technology has been widely used in biomedical experimental research and treatment of various diseases.

Cell microparticle

In the present invention, the terms "cell microparticles", "microparticles", and "microvesicles" are used interchangeably.

Cell microparticles are membrane corpuscles with diameters between 30 and 1000 nm that are secreted by cells in the body under normal and pathological conditions. Natural small particles, including extracellular bodies, containing cell contents, surrounded by cell membrane-like membrane structures. (exosome) and shedding vesicle (shedding vesicle). Both in vivo and in vitro experiments have demonstrated that cell microparticles can be secreted by a variety of cells such as red blood cells, B cells, T cells, dendritic cells, mast cells, epithelial cells, and tumor cells. The cells encapsulate specific biologically active molecules such as proteins, mRNAs, etc. into the cell microparticles, and these bioactive molecules are transported to the corresponding recipient cells through the cell microparticles. Regulating the biological function of the recipient cells, this cell-microparticle-mediated intercellular communication plays a very important role in some physiological and pathological processes.

The microparticles of the present invention include natural biovesicles having a lipid bilayer membrane secreted by cells, each having a size between 10 and 500 nm, including exosome, shedding vesicle, and A special name for shedding vesicles secreted by various cells.

Kit

The present invention provides a kit for promoting the transformation of somatic cells and/or primary competent stem cells into pluripotent stem cells, the kit comprising:

(i) a container, and an exogenous small RNA molecule (preferably in the form of a microparticle preparation or a first transfection reagent) located in the container;

(ii) a label or instructions indicating that the kit is for promoting the conversion of somatic cells and/or multipotent stem cells to pluripotent stem cells.

In a preferred embodiment, the kit further comprises (iii) a pH acidity regulator (second transfection reagent) for providing an acidic pH having a pH of from 2.5 to 6.0.

In a preferred embodiment, the pH adjusting agent is an acid.

In a preferred embodiment, the small RNA molecule comprises from 3 to 100, preferably from 4 to 50, more preferably from 5 to 20.

In a preferred embodiment, the small RNA molecules are placed separately or in combination.

In another preferred embodiment, the first transfection reagent and the second transfection reagent are separate reagents or two-in-one or the same transfection reagent.

The main advantages of the invention include:

(a) The method of the present invention increases the efficiency of small RNA transfected cells by adjusting the cell culture environment to be acidic.

(b) The method of the present invention has low toxicity, high transfection rate, and high stability of introduced small RNA molecules.

(c) The method of the present invention is convenient and efficient to operate, greatly simplifying the time and steps of laboratory operations.

(d) The present inventors have found for the first time that under the action of acid, small RNA molecules can regulate at least three pluripotent stem cell transcription factors and promote the transformation of somatic cells and/or multipotent stem cells into pluripotent stem cells.

(e) The present inventors have found for the first time that small RNA molecules can regulate at least three pluripotent stem cell transcription factors and promote the transformation of somatic cells into pluripotent stem cells under the guidance of cell microparticles.

The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Percentage and number of copies unless otherwise stated By weight.

Example 1 Introduction of osa-miRNA156a into wild-type 293T cells

In this embodiment, the experimental method is as follows:

1) PDL package

Formulate 250ml of 0.01% polylysine:

The container and operating equipment were sterilized, and polylysine was purchased from sigma.

A small amount of PBS was pipetted into a small plastic bottle of polylysine, and then the dissolved polylysine was added to about 200 ml of PBS.

Finally, add PBS to 250ml, filter and sterilize in a small bottle, store at 4 ° C for use, long-term should be stored at -20 ° C.

2) Paving

Using a 12-well plate, add 0.01% polylysine into each well, 3-4 drops per well. After all the additions, cover the 12-well plate and place at 37 ° C, 5% CO ₂ Incubate for 2-4 h in the incubator.

After the incubation, the 12-well plate was taken out and the polylysine was aspirated with a pipette. Change the straw, add PBS, 3-4 drops per well, rinse three times. Repeat the above steps three times.

The 12-well plate was placed in the incubator for use.

3) Cell culture

Placing 293T cells into two 12-well plates

Culture conditions: DMEM medium (10% FBS, 1% double antibody), cultured at 37 ° C, 5% CO 2 for 24 h.

4) Preparation of acidic culture solution

First, the DMEM culture solution (2% FBS, 1% double antibody) used in the experiment was prepared, and the pH was determined to be 7.65, and then the culture solution was adjusted to a pH of 3.15 with hydrochloric acid for use.

5) Transfer of nucleic acids

The transferred nucleic acid was selected from the exogenously synthesized rice single-stranded microRNA, osa-miR156a (synthesized by Gima, sequence UGACAGAAGAGAGUGAGCAC (SEQ ID NO.: 12)), and the rice osa-miR156a differed greatly from the transferred human 293T cells. It can effectively avoid the interference of own microRNA in animal cells and reduce the possibility of error in the experimental results.

To the culture solution of pH 7.65 and pH 3.15, osa-miR156a was added to a final concentration of 40 pmol/ml.

The 12-well plate with 293T cells was taken out from the incubator, the original culture solution was removed, and the above two pH-valued culture solutions supplemented with exogenous miRNA were added to the two 12-well plates, and incubated at 37 ° C for 0.5 h.

During the above incubation, RNase digest was prepared: RNase A (purchased from Thermo) was added to DMEM medium (2% FBS, 1% double antibody) in a volume ratio of 2 μl/ml, ie >100 u/ml.

After the above incubation time, the two pH-added culture solutions with exogenous miRNA were removed, washed once with PBS, and the prepared RNase digest was added, incubated at 37 ° C for 1 h, and then washed three times with PBS.

Cells were lysed with TRI Regent (purchased from Sigma) and total RNA was extracted as follows:

Cleavage for 5 min at room temperature; add 1/5 volume of chloroform, shake vigorously, let stand for 5 min at room temperature; centrifuge at 16000 g for 15 min at 4 ° C; carefully aspirate the supernatant, add 2 volumes of isopropanol, mix, -20 ° C Set 1h or overnight; 16000g, 4 ° C, centrifugation for 15min; discard the supernatant, add 1ml 75% ethanol (DEPC water preparation) to wash the precipitate, 16000g, 4 ° C, centrifugation for 15min; pour off the supernatant, after the precipitate is dried, The pellet was dissolved by adding an appropriate amount of DEPC water; OD260/OD280 was calculated to identify total RNA concentration and purity.

6) Detection of results

Quantitative detection of miRNA content in cells using qRT-PCR:

Real-time fluorescent quantitative PCR technology is to add a fluorescent group in the reaction system of PCR, and accumulate the fluorescent signal to monitor the whole PCR process in real time. In view of the length of miRNA 21 bp, a gene-specific reverse primer containing the same stem-loop structure was designed for each miRNA, and the specific cDNA was reversed, and finally PCR reaction was carried out.

The above-mentioned extracted RNA and other reagents required for the reaction are formulated into a reverse transcription reaction system, and the reaction is carried out under the following conditions, and the reverse transcription reaction conditions are as follows:

Step 1: 16 ° C 30 min

Step 2: 42 ° C 30 min

Step 3: 85 ° C 5 min

Step 4: Place at 4 ° C

After obtaining the corresponding cDNA according to the above reaction, it is combined with other reagents required for the reaction to form a PCR reaction system, and the reaction is carried out according to the following conditions to obtain a fluorescent signal value, and real-time fluorescent quantitative PCR reaction conditions are as follows:

Step 1: 95 ° C 5 min

Step 2: 95 ° C 15s

Step 3: 60 ° C 1 min

Step 2 - Step 3: 50 cycles

The processing of the data uses a relative comparison method and is also considered to be the ΔΔCt method. Ct is set to the number of cycles when the reaction reaches the domain value, and ΔCt = Ct sample - Ct internal reference. The expression level of the acid-treated exogenous microRNA relative to the control wild-type plant can be expressed by Equation 2 - ^ΔCT .

The results are as follows:

Figure 1 shows the relative values of two pH media transferred into the cells osa-miR156a, pH 7.65 control group The relative value of 1 showed that the exogenous microRNA content in the cells after pH3.15 transformation was significantly higher than that in the pH7.65 control group.

Example 2 by Lipofectamine ^TM 2000 and FAM siRNA transfection acid-induced

In this embodiment, the experimental method is as follows:

1) Lipofectamine ^TM 2000 transfection

Lipofectamine ^TM 2000, available from Invitrogen, FAM siRNA available from Zimmer, FAM siRNA with a fluorophore, after transfection by fluorescence microscopy, confocal microscopy or flow cytometry for detection. The operation steps are carried out according to the instructions. The approximate steps are as follows.

The cells were inoculated to a 12-well plate one day before transfection, and 400 μl of DMEM medium containing 10% calf serum (containing no double antibody) was added to each well, and the cell density should be 90% when transfected.

4-6 hours before replacing transfection serum-free medium (Opti-MEMΙ), After culturing for 4-6 hours with Lipofectamine ^TM 2000 siRNA transfected into the cell, particularly into the next step:

Dilute 100 pmol of siRNA into 45 μl of Opti-MEM® and mix gently.

Mix gently and Lipofectamine ^TM 2000, and then take Opti-MEMΙ 1μl into 49μl of diluted, mixed gently, incubated at room temperature for 5 minutes.

The diluted Lipofectamine ^TM 2000 siRNA and mixed gently and incubated at room temperature for 20 minutes to form a complex.

100 μl of the complex was added to each well, and the plate was translated back and forth, left and right to mix the complex with the cells.

4 to 6 hours after transfection (this time can be extended appropriately) to be replaced with serum-containing medium.

2) Acid-induced transfection of FAM siRNA

The method of acid-induced transfection of FAM siRNA was the same as that described in Example 1.

3) Detection of transfection efficiency

FAM is a green fluorescent group excited by blue light with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

Lipofectamine ^TM 2000 can be detected after 6 hours of transfection, and the cell treatment process before detection needs to be protected from light. For detection, the light path can be aligned with the hole that is not transfected with FMA siRNA, and the focal length is adjusted to turn on the excitation light. The observation time should not be too long to avoid the fluorescence being quenched.

Successfully transfected cells can be seen in FAM green fluorescence, which is dispersed in the cytoplasm.

The results are as follows:

Figure 2 is a schematic diagram showing the results of fluorescence induction of Lipofectamine ^TM 2000 and acid-induced transfection of FAM siRNA, showing that the transfection efficiency is comparable, indicating that the acid-treated group also has the ability to efficiently transfect siRNA.

Example 3 fetal bovine serum is rich in miRNA

The expression profile of fetal bovine serum was detected by solexa sequencing. The specific steps of solexa sequencing include:

(1) collecting fetal bovine serum samples;

(2) extracting total RNA from the sample by Trizol reagent;

(3) Performing PAGE electrophoresis to recover 17-27 nt RNA molecules;

(4) ligating an adaptor primer to the 3' and 5' ends of the small RNA molecule;

(5) The purified DNA was directly used for cluster generation and subjected to sequencing analysis using an Illumina Genome Analyzer.

The results showed that a large number of highly expressed animal miRNAs ("100 copies / 5 ml) were present in fetal bovine serum, including miR-432, miR-320, miR-27b, miR-103, miR-423, miR-185, miR-378. , miR-130b, let-7g, miR-107.

Previously, there have been few studies on miRNAs in fetal bovine serum in stem cells. During the culture of fetal bovine serum, these circulating miRNAs, exogenous small RNAs, are likely to be absorbed into cells and tissues.

Example 4 Circulating miRNA Regulating Transcription Factors of Pluripotent Stem Cells

Bioinformatics methods are used to predict miRNAs that regulate pluripotent stem cells.

The results indicate that 243 microRNAs may be involved in the regulation of transcription factors of pluripotent stem cells.

Among them, miRNAs that promote iPSC include: miR-291-3p, miR-294, miR-295, miR-302b, miR-372, miR-200c, miR-302s, miR-369s, miR-302a-3p, miR- 302b, miR-302c, miR-302d, miR-369-3p, miR-369-5p, miR-21, miR-214; miRNAs that promote Wisp1 include: miR-486; miRNAs that promote p21 include: miR-423- 5p; miRNAs that promote AOF1 include: miR-320a, miR-130; miRNAs that promote MECP2-1 include: miR-185, miR-432, miR-107, miR-103, miR-378, miR-27b, miR- 423-3p, miR-877, let-7g.

The transcription factors regulating iPSC include: Oct4, MET, Sox2, c-Myc, n-Myc, Klf4, Nanog, Lin28, AOF1, AOF2, MECP1-p66, MECP2, MBD2, p21, p53, Wisp1, ZEB2, Jun.

It is predicted by bioinformatics that miRNAs rich in fetal bovine serum can inhibit genes such as p21 and p53 that can prevent somatic cells from becoming pluripotent stem cells, suggesting that miRNAs rich in fetal bovine serum may induce somatic cells to become Multifunctional stem cells.

Since then, the inventors have found through extensive research that miRNAs which are expressed in fetal bovine serum are also present in stem cells. 11 kinds of miRNAs highly expressed in stem cells were screened by extensive screening, and the results are shown in Table 1.

Table 1

Example 5 Preparation of pluripotent stem cells by exogenous small RNA

The pluripotent stem cells are efficiently prepared by stimulating exogenous small RNA to induce the transformation of somatic cells into pluripotent stem cells by acid-mediated, cell microparticle-mediated, plasmid-mediated, and the like.

5.1 Acid-mediated exogenous small RNA preparation of pluripotent stem cells

CD45 ⁺ hematopoietic stem cells were isolated from mice by flow cytometry, and 10 ⁶ CD45 ⁺ hematopoietic stem cells were transferred to HBSS culture medium containing 20% fetal bovine serum (pH = 5.7 with hydrochloric acid), and cultured at 37 ° C for 25 min. It was then centrifuged at 1000 rpm for 5 min at room temperature. Extract RNA from some cells and use qRT-PCR to detect whether the miRNAs in fetal bovine serum that may induce somatic cells to become pluripotent stem cells enter the cells under the treatment of acid (such as phosphoric acid, sulfuric acid, or acetic acid). .

Another part of the cells was colonized in a Petri dish without cell adhesive, and cultured in a DMEM/F12 medium supplemented with 1000 U of Leukemia Inhibitor Factor (LIF) and 2% B27, and some cells were separated every day for 1-10 days. The expression of pluripotent stem cell-specific genes such as OCT4 was examined, and the induction rate of pluripotent stem cells was observed and counted.

The results indicate that acid-mediated exogenous small RNA can regulate the expression of pluripotent stem cell-specific genes and significantly increase the induction rate of pluripotent stem cells.

5.2 Preparation of pluripotent stem cells by stimulation of exogenous small RNA by plasmid transfection

CD45 ⁺ hematopoietic stem cells were isolated from mice by flow cytometry, and 10 ⁶ CD45 ⁺ hematopoietic stem cells were transferred to DMEM/F12 medium containing 1000 U of Leukemia Inhibitor Factor (LIF) and 2% B27, followed by utilization. Liposomes are loaded into cells by plasmids that are capable of overexpressing miRNAs in fetal calf serum that may induce somatic cells to become pluripotent stem cells, followed by isolation of some cells such as OCT4 every day for 1-10 days. The expression of functional stem cell-specific genes was observed and the induction rate of pluripotent stem cells was observed and counted.

In the present invention, the plasmid expression vector of the miRNA of pluripotent stem cells is not particularly limited, and includes an expression vector which is commercially available or prepared by a conventional method. Representative examples include, but are not limited to, pcDNATM6.2-GW/miR, pcDNA3, pMIR-REPORT miRNA, pAdTrack-CMV, pCAMBIA3101+pUC-35S, pCMVp-NEO-BAN, pBI121, pBin438, pCAMBIA1301, pSV2 , CMV4 expression ^{vectors, pmiR-RB-Report TM,} pshOK-basic, mmu-mir 300-399miRNASelect TM, pshRNA-copGFP Lentivector, GV317, GV309, GV253, GV250, GV249, GV234, GV233, GV232, GV201, GV159 , or other GV series expression vector.

The results indicate that plasmid-mediated exogenous small RNA can regulate the expression of pluripotent stem cell-specific genes and significantly increase the induction rate of pluripotent stem cells.

5.3 Preparation of pluripotent stem cells by stimulating exogenous small RNA mediated by microparticles

Microvesicles (MV) in fetal bovine serum were separated by ultracentrifugation (100000 g, 70 min). The miRNA in the MV was then detected using qRT-PCR technology. The specific steps of qRT-PCR include:

Take 100 μl of MV in serum, add 300 μl of DEPC water, mix and add 200 μl of water-saturated phenol, add 200 μl of chloroform after vigorous shaking, shake well, centrifuge at 16000 g for 15 minutes at room temperature; carefully pipette after centrifugation The supernatant (about 400 μl), add 2 volumes of isopropanol, let stand at -20 ° C for 60 minutes, then 16000 g, centrifuge at 20 ° C for 20 minutes; after centrifugation, discard the supernatant, add 75% ethanol, gently invert, 16000 g, centrifuged at 4 ° C for 20 minutes; after centrifugation, discard the supernatant and add 20 μl of DEPC water to dissolve. Two microliters of RNA was taken for reverse transcription, and then 1 μl of cDNA was taken for real-time PCR to detect the expression of the target small nucleic acid.

The primary cells were cultured to a suitable density, and microvesicles (MV) in fetal bovine serum were added to the medium, incubated for 10-30 min under cell culture conditions, and cultured continuously and observed until stem cells were confirmed to be present. The cells were isolated to detect the expression of pluripotent stem cell-specific genes such as OCT4, and the induction rate of pluripotent stem cells was observed and counted.

The results indicate that miRNAs enriched in fetal bovine serum that may induce somatic cells to become pluripotent stem cells are present in the MV. Cellular microparticles mediated stimulation of fetal bovine serum miRNA promotes the transformation of somatic cells into pluripotent stem cells.

The invention also increases the expression level of exogenous small RNA or stimulates small RNA to promote the transformation of somatic cells into pluripotent stem cells, and further comprises calcium phosphate coprecipitation, electroporation, DEAE-dextran and polybrene, mechanical method (microinjection) And gene gun), cationic liposome reagent transfection method, and the like.

Example 6 Acid-stimulated exogenous small RNA was introduced into somatic cells to efficiently prepare pluripotent stem cells

6.1. Acid-stimulated exogenous siRNA is introduced into somatic cells to efficiently prepare pluripotent stem cells

First, 3 generations of 12-well plates were taken for 3 generations of OG2-MEF cells (obtained from the Life Science Institute of Nanjing University) 3×10 ⁴ /well, and 12 hours later, retrovirus was induced by Oct4, Klf4, and Sox2. Infection, culture in MES+Vc medium, cultured at 37 ° C, 5% CO _{2 for} 24 h.

Exogenous siRNA was selected for subsequent experimental studies. The sequence information is shown in Table 2. The first sequence of each gene was selected for the experiment.

Table 2

Sequence name Jun siRNA SEQ ID NO.: Numbering Justice chain (5'-3') 1 CAGUAACCCUAAGAUCCUAAA dTdT 13 2 GCUAACGCAGCAGUUGCAAAC dTdT 14 3 GGAUCGCUCGGCUAGAGGAAA dTdT 15 Sequence name Mbd2 siRNA Numbering Justice chain (5'-3') 1 GGAAACAGGAAGAGCGAGU dTdT 16 2 AGGAGGAAGUGAUCCGAAA dTdT 17 3 GCAAGAUGAUGCCUAGUAA dTdT 18 Sequence name P53 siRNA Numbering Justice chain (5'-3') 1 GAAUGAGGCCUUAGAGUUA dTdT 19 2 GGGACAGCCAAGUCUGUUA dTdT 20 3 CCGCCGUACAGAAGAAGAA dTdT twenty one

The primer sequences of the Jun siRNA, Mbd2 siRNA, and p53 siRNA were as described in Table 3.

table 3

Sequence name Primer sequence (5'-3') SEQ ID NO.: jun-si-1-F ACACTCCAGCTGGGCAGTAACCCTAAGAT twenty two p53-si-1-F ACACTCCAGCTGGGGAAUGAGGCCUUAGA twenty three MBD2-si-1-F ACACTCCAGCTGGGGGAAACAGGAAGAGC twenty four

Then proceed to the experimental grouping (1):

 RNAiMAX转染。Transfection is used as a transfection group.

RNAiMAX transfection.

Lipofectamine ^TM RNAiMAX were purchased from Invitrogen, FAM siRNA available from Zimmer, FAM siRNA with a fluorophore, after transfection by fluorescence microscopy, confocal microscopy or flow cytometry for detection. The operation steps are carried out in accordance with the instructions, and the steps are as described in Embodiment 2.

Group T: (initial cell density 2 × 10 ⁴ ) See Table 4.

Table 4

OKS T-3si T+jun-si OKS T-3si T+jun-si OKS T-3si T+jun-si DR

Note: OKS represents a positive control group, and normal induction is not treated;

DR represents a negative control group, only infected with DsRed virus for estimating infection efficiency, and does not induce positive clones;

T-3si represents the siRNA concentration of jun, mbd2 and p53 genes transfected to 10nM/25nM

T+Jun-si represents a siRNA concentration of 10nM/25Nm for transfection-induced jun gene

Group A (Acid) was an acid-treated group (the cells were relatively dying due to acid treatment, and the number of cells was relatively high when plating), the acid pH was 3.5, and the acid treatment time was 15 min.

Group A: (Initial cell density 5.5 × 10 ⁴ ) See Table 5.

table 5

A A-3si A+jun-si A A-3si A+jun-si NA-3si A-3si A+jun-si DR

Note: NA-3si represents acid-free treatment, only the above three siRNAs are added;

A-3si represents acid-induced jun, mbd2, p53 three genes siRNA concentration of 10nM/25nM;

A+Jun-si represents the acid-induced jun gene siRNA concentration of 10nM/25nM;

The sequence information is the same as the T group.

Both group T and group A were treated every two days from the next day to the eighth day. Positive clones were counted on the ninth day, and the number of induced stem cell clones that emitted green fluorescence was manually counted under a fluorescence microscope.

The specific results are as follows:

The results of Day 9 positive clones are shown in Table 6.

Table 6

Group T OKS T+3si T+Jun-si 145 773 351 172 716 412

For the transfection group, compared with the control group (OKS), the number of positive clones was significantly increased by siRNA targeting jun, mbd2, and p53, and the number of positive clones transfected with siRNA specifically inhibiting jun gene. increase. The above results indicated that in the induction process, siRNAs of jun, mbd2 and p53 were transfected with liposomes, and the expression of these three genes was inhibited, which promoted the efficiency of inducing stem cells. Transfection of jun gene siRNA with liposome inhibits the expression of jun gene and promotes the efficiency of inducing stem cells.

The results of group A are shown in Table 7.

Table 7

Group A A A+3si A+Jun-si NA+3si 200 337 268 194 202 330 266

Simple homogenization was performed using the number of plated cells, and the results of positive clones are shown in Table 8.

Table 8

Group A A A+3si A+Jun-si NA+3si 73 123 97 71 73 120 97

In the acid-induced group, compared with the control group (A, NA+3si), the number of positive clones of siRNA, mbd2, p53, and the number of positive clones increased; the number of positive clones of acid-induced jun gene increased. It is indicated that by directly adjusting the pH of the medium to pH 3.5, siRNAs of jun, mbd2 and p53 genes can be induced to enter cells, thereby inhibiting the expression of these three genes and improving the efficiency of inducing stem cells; The medium pH is temporarily adjusted to pH 3.5 for induction, which can promote the jun gene siRNA to enter the cell, exert the effect of inhibiting the expression of the jun gene, and improve the efficiency of inducing stem cells.

Experimental group (2):

The T group (Transfection) was the transfection group, the group A (Acid) was the acid treatment group, and the acid treatment time was 15 min;

OKS: positive control group, normal induction is not treated;

P53-si: siRNA 10nM/25nM transfected/acid-induced p53 gene;

Both group T and group A were treated every two days from the next day to the eighth day. Positive clones were counted on the ninth day, and the specific results are shown in Table 9.

Table 9

For the transfection group, the number of positive clones that induced siRNA that specifically inhibits the p53 gene was increased as compared with the control group (OKS). It is indicated that siRNA transfected with p53 gene by liposome can inhibit the expression of p53 gene and promote the efficiency of inducing stem cells. In the acid-induced group, the number of positive clones of siRNA-induced p53 gene siRNA increased compared with the control group (OKSA, NA+3si), indicating that induction by temporarily adjusting the pH of the medium to pH 3.5 can promote The siRNA of the p53 gene enters the cell, exerts an effect of inhibiting the expression of the p53 gene, and increases the efficiency of inducing stem cells.

Acid-stimulated other exogenous siRNAs are introduced into somatic cells, and pluripotent stem cells can be efficiently prepared. Other siRNAs include: Zeb2 siRNA, Mecp2 siRNA, p21 siRNA, and wisp1 siRNA. The sequence information of other siRNAs is shown in Table 10.

Table 10

Sequence name Zeb2 siRNA SEQ ID NO.: Numbering Justice chain (5'-3') 1 GAAGGAAGAUUAUGAUGCA dTdT 25 2 CAUCAGACUUUGAGGAAUA dTdT 26 3 GCAUAAACUUAGACCACAA dTdT 27 Sequence name Mecp2 siRNA Numbering Justice chain (5'-3') 1 GUCCACCCUUGGUGAGAAA dTdT 28 2 GCUCUAAAGUAGAAUUGAU dTdT 29 3 GCUGGAAAGUAUGAUGUAU dTdT 30 Sequence name P21 siRNA Numbering Justice chain (5'-3') 1 GAACAUCUCAGGGCCGAAA dTdT 31 2 CCAGCCUGACAGAUUUCUA dTdT 32

3 GAGAACGGUGGAACUUUGA dTdT 33 Sequence name Wisp1 siRNA Numbering Justice chain (5'-3') 1 GCGUCAGCCUAAUCACAGA dTdT 34 2 CAACCCAACUGCAGGUACA dTdT 35 3 CCACUAGAGGAAACGACUA dTdT 36

6.2. Acid-stimulated exogenous miRNAs are introduced into somatic cells to efficiently prepare pluripotent stem cells

First, 3 generations of 12-well plates were taken for 3 generations of OG2-MEF cells (obtained from the School of Life Sciences, Nanjing University) 3×10 ⁴ /well, and 12 hours later, retrovirus was induced by Oct4, Klf4, and Sox2. Infection, culture in MES+Vc medium, cultured at 37 ° C, 5% CO _{2 for} 24 h.

Then miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369-3p, miR-369-5p, miR-21, miR-214 were selected for group experiments. Each miRNA was transfected with liposome and acid-treated, and the concentration of each miRNA was 10 nM/25 nM. The sequence information of miRNAs is shown in Table 11.

Table 11

Sequence name Sequence information SEQ ID NO.: mmu-miR-302a-3p Aagugcuuccauguuuuaguga 37 mmu-miR-302b Uaagugcuuccauguuuuaguag 38 mmu-miR-302c Uaagugcuuccauguuucagugg 39 mmu-miR-302d Uaagugcuuccauguuuuagu 40 mmu-miR-369-3p Aauaauacaugguugaucuuu 41 mmu-miR-369-5p Aucgaccguguuauauucgc 42 mmu-miR-21 Uagcuuaucagacugauguuga 43 mmu-miR-214 Acagcaggcacagacaggcagu 44

The T group (Transfection) was the transfection group, the group A (Acid) was the acid treatment group, and the acid treatment time was 15 min;

OKS: Positive control group, normal induction was not treated.

Both group T and group A were treated every two days from the next day to the eighth day. Positive clone counts were performed on the ninth day.

As a result of the analysis, transfection-inducible specific miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369-3p, miR- were compared with the control group (OKS). The number of positive clones of 369-5p, miR-21 and miR-214 increased. The use of liposome to transfect miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369-3p, miR-369-5p, miR-21, miR-214 for the induction of stem cells Efficiency has a positive effect. For the acid-induced group, acid-induced miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369-3p, miR-369-5p, miR- compared to the control group (OKSA) twenty one, The number of positive clones of miR-214 increased, indicating that the induction of miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369- can be induced by transiently adjusting the pH of the medium to pH 3.5. 3p, miR-369-5p, miR-21, and miR-214 enter the cell, play a role in inhibiting gene expression, and increase the efficiency of inducing stem cells.

Example 7 kit

A kit for promoting transformation of somatic cells and/or multipotent stem cells into pluripotent stem cells, the kit comprising:

(i) a container, and exogenous small RNA molecules located within the container;

(ii) a label or instructions indicating that the kit is for promoting the conversion of somatic cells and/or multipotent stem cells to pluripotent stem cells. with

(iii) a pH acidity regulator for providing an acidic pH having a pH of from 2.5 to 6.0.

Wherein, the small RNA molecule comprises from 3 to 100, preferably from 4 to 50, more preferably from 5 to 20, preferably one or more combinations of the miRNAs shown in Table 1. The exogenous small RNA molecules are placed separately or in combination.

The above kit can be used to regulate the expression of pluripotent stem cell transcription factors; and/or to promote the transformation of somatic cells and/or multipotent stem cells into pluripotent stem cells.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Claims (18)

Use of an exogenous small RNA molecule, characterized in that it is used for preparing a reagent or a kit for promoting transformation of a somatic cell or a multipotent stem cell into a pluripotent stem cell, wherein the reagent or kit comprises a transfection reagent, The transfection reagent includes a pH acidity regulator. The use according to claim 1, wherein the transfection reagent is a microparticle preparation. The use according to claim 1, wherein the small RNA molecule comprises at least two or all of the miRNAs selected from the group consisting of (A): miR-432, miR-320, miR-27b, miR-103. The use according to claim 3, wherein the small RNA molecule further comprises at least one or all of the miRNAs selected from the group consisting of: miR-423, miR-185, miR-378, miR-130b . The use according to claim 3 or 4, wherein the small RNA molecule further comprises at least one or all of the miRNAs selected from the group (C): let-7g, miR-107. The use according to claim 3 or 4 or 5, wherein the small RNA molecule comprises ≥ 4 selected from the group (A), the group (B) and/or the group (C), preferably ≥ Six species, more preferably > 7 species, optimally > 8 or all miRNAs. The use according to claim 3, wherein the small RNA molecule comprises at least 3 or all miRNAs selected from the group (A). The use according to any one of claims 1 to 7, wherein the small RNA molecule comprises at least four or all of the miRNAs selected from the group consisting of: (h): hsa-miR-432-5p, hsa- miR-320a, hsa-miR-27b-3p, hsa-miR-103a-3p, hsa-miR-423-5p, hsa-miR-185-5p, hsa-miR-378a-3p, hsa-let-7g- 5p, hsa-miR-130b-3p, or a combination thereof. The use according to any one of claims 1 to 8, wherein the small RNA molecule comprises at least four or all miRNAs selected from the group consisting of (b): bta-miR-107, bta-miR- 432, bta-miR-320a, bta-miR-27b, bta-miR-103, bta-miR-423-3p, bta-miR-423-5p, bta-miR-185, bta-miR-378, bta- Let-7g, bta-miR-130b, or a combination thereof. The use according to any one of claims 1 to 9, wherein the small RNA molecule comprises at least four or all miRNAs selected from the group consisting of: (f) mmu-miR-320-3p, mmu- miR-27b-3p, mmu-miR-103-3p, mmu-miR-423-5p, mmu-miR-185-5p, mmu-let-7g-5p, mmu-miR-130b-3p, or a combination thereof. The use according to any one of claims 1 to 10, wherein the small RNA molecule comprises at least one or all of the miRNAs selected from the group consisting of (G): miR-302a-3p, miR-302b, miR-302c, miR-302d, miR-369-3p, miR-369-5p, miR-21, miR-214, or a combination thereof. An in vitro method for promoting transformation of somatic cells or multipotent stem cells into pluripotent stem cells, comprising the steps of: (i) providing a transfection system comprising: (a) a buffer and/or a culture; (b) somatic or multipotent stem cells; and (c) small RNA; (ii) placing the transfection system under transfection conditions such that the small molecule RNA is transfected into the somatic or multipotent stem cells; (iii) inducing culture of the transfected somatic cells or multipotent stem cells obtained in the previous step, thereby converting the somatic cells or the multipotent stem cells into pluripotent stem cells; Wherein the transfection conditions comprise an acidic pH treatment. The method of claim 12 wherein said acidic pH treatment is carried out at a pH of from 2.5 to 6.0, preferably from 2.8 to 5.5, more preferably from 2.9 to 4.5, most preferably from 3.0 to 4.0. The method according to claim 12 or 13, wherein the acidic pH treatment is carried out for a period of from 0.5 minutes to 24 hours. The method according to claim 12, wherein in the step (iii), the transfected somatic or multipotent stem cells obtained in the previous step are transferred to the somatic cells of the exogenous nucleic acid, And induction is performed to obtain pluripotent stem cells. A composition or combination for promoting the conversion of somatic or multipotent stem cells into pluripotent stem cells, comprising: (i) an exogenous small RNA molecule; and (ii) a pH acidity regulator for providing an acidic pH having a pH of from 2.5 to 6.0. Use of a composition or combination according to claim 16 for the preparation of a kit for promoting the conversion of somatic cells and/or multipotent stem cells to pluripotent stem cells. A kit, characterized in that the kit comprises: (i) a container, and exogenous small RNA molecules located within the container; (ii) a label or instructions indicating that the kit is for promoting the conversion of somatic cells and/or multipotent stem cells to pluripotent stem cells; (iii) a pH acidity regulator for providing an acidic pH having a pH of from 2.5 to 6.0.

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