A reprogrammed responsive intelligent hydrogel material and its preparation method and application

Abstract

The present invention relates to a reprogramming responsive intelligent hydrogel material and a preparation method and application thereof. The hydrogel material provided by the present invention uses alginate as a skeleton, and uses RGD series polypeptides and polyglutamic acid to modify the skeleton respectively. The iPS cell line obtained by using the hydrogel material provided by the present invention to mediate somatic cell reprogramming is similar to embryonic stem cells, showing high expression of pluripotency-related genes at the transcriptional level and translational level, strong positive staining of alkaline phosphatase, and demethylation patterns of promoter regions of endogenous pluripotency-related genes Oct4 and Nanog. The hydrogel material provided by the present invention can quickly and efficiently obtain individual-specific induced pluripotent stem cells, laying a foundation for the acquisition of individual-specific induced pluripotent stem cells and their wide application in the fields of regenerative medicine, disease simulation and drug screening.

Description

Technical Field

[0001] The present invention relates to the fields of biomedical engineering and pluripotent stem cells. Specifically, the present invention relates to a reprogrammed responsive intelligent hydrogel material and a preparation method and application thereof. Background Art

[0002] Embryonic stem cells are a type of pluripotent cells derived from the inner cell mass of the early embryo during the development of the fertilized egg. They have the potential for unlimited proliferation and multidirectional differentiation, and in theory can differentiate into almost all types of terminally differentiated cells in the human body. Therefore, embryonic stem cells have broad application prospects in the fields of basic research in developmental biology and regenerative medicine. The source and ethical issues of human embryonic stem cells, especially patient-specific stem cells, have become a difficult problem that has plagued the scientific and translational communities, and have largely limited the widespread application of embryonic stem cells in the fields of basic research and translational research. Therefore, it is a very important scientific issue to use a rich source of various types of somatic cells and use cell reprogramming technology to enable terminally differentiated somatic cells to regain induced pluripotent cells similar to embryonic stem cells.

[0003] There are currently three main ways to obtain pluripotent stem cells: somatic cell nuclear transfer (SCNT), cell fusion, and induced pluripotent stem cell reprogramming (iPS). A large number of studies have shown that induced pluripotent stem cells (iPS cells) are very similar to embryonic stem cells and can form chimeric mice with germline transmission ability after being injected into the blastocyst of a recipient mouse. In 2009, the research team of Academician Zhou Qi and the research team of Professor Gao Shaorong from China respectively proved the pluripotency of induced pluripotent stem cells (referred to as iPS cells in this patent) through tetraploid compensation technology. In 2007, the Yamanaka laboratory and the Thompson laboratory successfully induced human skin cells into iPS cells (Takahashi et al., 2007; Yu et al., 2007).

[0004] In general, the induced pluripotent stem cell reprogramming technology, through the introduction of exogenous factors, causes terminally differentiated somatic cells to undergo cell reprogramming and obtain induced pluripotent stem cells. This technology is a milestone in the field of stem cell research and developmental biology research. This breakthrough technology can more easily obtain pluripotent stem cells derived from the patient's own body cells, which not only solves the problem of cell sources in regenerative medicine, but also avoids xenogeneic and allogeneic immune rejection, circumvents ethical constraints, and lays a solid foundation for regenerative medicine, disease simulation and drug screening.

[0005] As a new technology, many aspects of induced pluripotent stem cell technology need further in-depth research. For example, the molecular mechanism of obtaining iPS cells through cell reprogramming is not fully understood; the process of inducing iPS cells is inefficient; the process of inducing iPS cells is time-consuming and faces potential safety risks in translational applications.

[0006] The efficiency of conventional iPS induction is very low, with only about 0.01%-1% of the starting cells being able to complete the cell reprogramming process and obtain iPS cells. Therefore, scientists have adopted a variety of methods to improve the efficiency of inducing iPS cells. For example, adding small molecule compounds, such as Vc, can significantly improve the efficiency of obtaining iPS cells. Other research works have fused the transcription activation domain (TAD) of VP16 or YAP with related transcription factors to form genetically engineered transcription factors, which greatly improves the efficiency of inducing iPS cells. In addition, the cycle for inducing mouse iPS cells is three to four weeks, while the cycle for inducing human iPS cells is relatively longer.

[0007] In general, the iPS research field has made many breakthroughs in recent years, reflecting the broad prospects of iPS cells in regenerative medicine, disease simulation and drug screening. iPS technology still has scientific problems such as long induction time and low induction efficiency. Therefore, being able to induce iPS cells quickly and efficiently will greatly promote regenerative medicine, disease simulation and drug screening.

[0008] In recent years, the role of biomechanical factors in the regulation of pluripotency has attracted increasing attention from researchers. Cell reprogramming is a highly dynamic and complex process. At present, there is no reprogramming-responsive smart biomaterial that can truly sense the dynamic changes of the cell reprogramming process and change accordingly with the changes in the cell reprogramming process. The reprogramming process is a dynamic and complex process, which includes the reorganization of the cytoskeleton, the remodeling of the metabolic mode, and the changes in extracellular pH during the reprogramming process. Therefore, designing smart hydrogel materials that can sense the reorganization of the cytoskeleton and the remodeling of the metabolic mode-the changes in extracellular pH during cell reprogramming, and quickly and efficiently obtaining high-quality iPS cells is an unresolved scientific problem. Summary of the invention

[0009] The purpose of the present invention is to improve the efficiency of somatic cell reprogramming to generate induced pluripotent stem cells (iPS).

[0010] In order to solve the problems of low efficiency and long time in inducing iPS in the iPS research field, the present invention proposes a new concept of reprogramming responsive intelligent hydrogel material, designs a reprogramming responsive intelligent hydrogel material, and provides a method for obtaining iPS cells by mediating cell reprogramming with the reprogramming responsive intelligent hydrogel material.

[0011] In a first aspect, the present invention provides a reprogrammed responsive intelligent hydrogel material, wherein the hydrogel material uses alginate as a skeleton, and the skeleton is modified by chemical molecules, wherein the chemical molecules are RGD series polypeptides and polyglutamic acid.

[0012] Furthermore, the present invention uses the reprogramming responsive intelligent hydrogel material as a matrix material for mediating the cell reprogramming process.

[0013] In a second aspect, the present invention provides a method for preparing a reprogrammed responsive intelligent hydrogel material, comprising: using alginate as a skeleton material, performing a carboxyl activation reaction on the side chains of alginate, and then using an amino reaction to modify the RGD series polypeptides and polyglutamic acid to the side chains of the alginate skeleton material, respectively, to form a reprogrammed responsive intelligent hydrogel material.

[0014] According to the understanding of those skilled in the art, the present invention also seeks to protect the use of the above-mentioned reprogramming-responsive intelligent hydrogel material in mediating somatic cell reprogramming to obtain induced pluripotent stem cells.

[0015] In a third aspect, the present invention provides a method for obtaining induced pluripotent stem cells by reprogramming somatic cells, using the above-mentioned reprogramming-responsive intelligent hydrogel material as an induction medium. Specifically, somatic cells undergoing reprogramming are inoculated into the reprogramming-responsive intelligent hydrogel material, and induced pluripotent stem cells are finally obtained. Moreover, in the method provided by the present invention, somatic cell reprogramming is mediated by proteins encoded by pluripotency-related genes.

[0016] More specifically, in the method provided by the present invention, the pluripotency-related genes include but are not limited to SOX2, Sall4, OCT4, Zfp42, Nr5a2, Daxl, Eras, Esrrb, Dppa2, Lin28A, Utf1, Klf4, Glis1, Prdm1, Prdm14, c-myc, and Tbx5; preferably, the pluripotency-related genes are selected from one of Sox2, Oct4, c-myc and Klf4.

[0017] In the method provided by the present invention, the nucleotide sequences of the pluripotency-related genes are shown in SEQ ID NO.1 (Oct4), SEQ ID NO.3 (Klf4), SEQ ID NO.5 (Sox2), SEQ ID NO.7 (c-myc) and SEQ ID NO.9 (Glis1).

[0018] In the method provided by the present invention, the protein is one or more of the proteins encoded by the Sox2, Oct4, c-myc and Klf4 genes of mice, rats, pigs, cattle, crab-eating monkeys or humans; preferably, the amino acid sequence of the protein is shown in SEQID NO.2 (Oct4), SEQ ID NO.4 (Klf4), SEQ ID NO.6 (Sox2), SEQ ID NO.8 (c-myc) and / or SEQID NO.10 (Glis1).

[0019] In the method provided by the present invention, the somatic cells are somatic cells of mammalian origin; preferably, the somatic cells include blood cells, skin cells, Sertoli cells and / or urine epithelial cells.

[0020] As a specific embodiment of the present invention, a method for obtaining induced pluripotent stem cells by reprogramming somatic cells comprises:

[0021] (1) using one or more of the pluripotency-related genes SOX2, Sall4, OCT4, Zfp42, Nr5a2, Daxl, Eras, Esrrb, Dppa2, Lin28A, Utf1, Klf4, Glis1, Prdm1, Prdm14, c-myc, and Tbx5 to construct expression vectors and treat somatic cells;

[0022] (2) The treated somatic cells obtained in step (1) are inoculated into a reprogramming responsive smart hydrogel material to screen and obtain induced pluripotent stem cells.

[0023] In the method provided by the present invention, the treatment is to introduce the nucleotide sequence, amino acid sequence or expression vector of the pluripotency-related gene into somatic cells; preferably, the treatment includes plasmid transfection, electrofection, viral infection, mRNA transfection and / or protein transduction.

[0024] The beneficial effects of the present invention are:

[0025] The reprogramming responsive smart hydrogel material provided by the present invention has a natural polysaccharide Alg as a skeleton, which has multiple functional groups such as polyhydroxyl and carboxyl groups distributed along the main chain and is easy to be chemically modified. In the present invention, the Alg skeleton is modified with RGD and PGA respectively to provide a "tailor-made" smart hydrogel material for cell reprogramming.

[0026] The cell reprogramming responsive smart hydrogel material of the present invention successfully induced various types of somatic cells to complete cell reprogramming and successfully obtained iPS cells, demonstrating that the reprogramming responsive smart hydrogel material of the present invention has powerful application scenarios.

[0027] The reprogrammed responsive intelligent hydrogel material of the present invention can quickly and efficiently obtain individual-specific induced pluripotent stem cells, laying a foundation for the acquisition of individual-specific induced pluripotent stem cells and their wide application in the fields of regenerative medicine, disease simulation and drug screening.

[0028] By using the reprogramming responsive hydrogel material provided by the present invention, iPS cells can be obtained efficiently and quickly when mediating somatic cell reprogramming.

[0029] The iPS cell line obtained by somatic cell reprogramming mediated by the reprogramming responsive hydrogel material provided by the present invention is similar to embryonic stem cells (ES), showing high expression of pluripotency-related genes at the transcriptional and translational levels, strong positive staining of alkaline phosphatase, and demethylation patterns of the promoter regions of endogenous pluripotency-related genes Oct4 and Nanog.

[0030] Furthermore, the method for obtaining induced pluripotent stem cells by somatic cell reprogramming provided by the present invention can achieve the characteristics of inducing embryoid bodies to differentiate into three different germ layers, producing tissues of three different germ layers through teratoma experiments and having normal karyotypes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the morphological characterization of the reprogrammed responsive intelligent hydrogel material in the present invention when forming stable hydrogel microspheres. A is the detection result of an optical microscope, and B is the detection result of a scanning electron microscope.

[0032] Figure 2It is a characterization of the characteristics of the reprogrammed responsive smart hydrogel material of the present invention. Wherein, A is the detection of the elastic modulus of the reprogrammed responsive smart hydrogel material; SA2.1G' and SA2.1G" in A represent the elastic modulus and viscous modulus of SA2.1, SA2.2G' and SA2.2G" represent the elastic modulus and viscous modulus of SA2.2, SA2.3G' and SA2.3G" represent the elastic modulus and viscous modulus of SA2.3, SA2.4G' and SA2.4G" represent the elastic modulus and viscous modulus of SA2.4, and SA3.1G' and SA3.1G" in A represent A is the elastic modulus and viscous modulus of SA3.1, SA3.2G' and SA3.2G" represent the elastic modulus and viscous modulus of SA3.2, SA3.3G' and SA3.3G" represent the elastic modulus and viscous modulus of SA3.3, and SA3.4G' and SA3.4G" represent the elastic modulus and viscous modulus of SA3.4; B is the detection of the elastic modulus of the reprogrammed responsive intelligent hydrogel material under different pH conditions; C is the detection of the chemical components of the reprogrammed responsive intelligent hydrogel material by Fourier infrared spectrometer.

[0033] Figure 3 The statistical results of stem cell clone size and alkaline phosphatase-positive clone number induced by reprogramming responsive smart hydrogel materials. In the figure, 2.1 represents a conventional three-dimensional material with a relatively hard hardness, 2.5 represents a conventional three-dimensional material with a relatively soft hardness, and 2.5-RL-PL represents a reprogrammed responsive smart hydrogel material.

[0034] Figure 4 The morphology and alkaline phosphatase expression of pluripotent stem cells (iPS) induced by the reprogramming responsive intelligent hydrogel material of the present invention are identified. Among them, A is the morphology of iPS cells, which is similar to the morphology of embryonic stem cells (ES). B is the positive staining of alkaline phosphatase in pluripotent stem cells (iPS) obtained by cell reprogramming mediated by the reprogramming responsive intelligent hydrogel material.

[0035] Figure 5 The expression of pluripotency-related marker genes in pluripotent stem cells (iPS) induced by the reprogramming responsive intelligent hydrogel material of the present invention at the mRNA level is detected. As shown in the figure, the endogenous pluripotency-related marker genes Esrrb (A), Utf1 (B), Oct4 (C) and Nanog (D) in the pluripotent stem cells (iPS) induced by the reprogramming responsive intelligent hydrogel material show significantly high expression.

[0036] Figure 6The expression of pluripotency-related marker genes in the pluripotent stem cells (iPS) induced by the reprogramming responsive smart hydrogel material of the present invention at the protein level is detected. As shown in the figure, the endogenous pluripotency-related marker genes Oct4 and Sox2 in the pluripotent stem cells (iPS) induced by the reprogramming responsive smart hydrogel material show significantly high expression.

[0037] Figure 7 The methylation status of the specific regions of the promoters of the pluripotency-related factors Oct4 and Nanog in the pluripotent stem cells (iPS) induced by the reprogramming responsive intelligent hydrogel material described in the present invention in different samples. As shown in the figure: A is a MEF sample; B and C are pluripotent stem cells (iPS) induced by the reprogramming responsive intelligent hydrogel material described in the present invention, recorded as 3D-iPS-1# and 3D-iPS-2# samples respectively; D is an embryonic stem cell (ESC) positive control sample.

[0038] Figure 8 This is the karyotype detection of pluripotent stem cells (iPS) induced by the reprogramming responsive intelligent hydrogel material described in the present invention.

[0039] Fig. 9 The pluripotent stem cells (iPS) induced by the reprogrammed responsive intelligent hydrogel material of the present invention can generate teratomas after being injected into recipient mice, and after HE staining, the structures of the three germ layers are displayed. DETAILED DESCRIPTION

[0040] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention all fall within the scope of protection of the present invention.

[0041] Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the embodiments of the present invention are all commercially available; unless otherwise specified, all technical means in the embodiments of the present invention are conventional means well known to those skilled in the art.

[0042] Example 1 Preparation method of hydrogel material

[0043] This embodiment provides a method for preparing a hydrogel material. The raw materials for preparing the hydrogel material in this embodiment include:

[0044] (1) Sodium alginate (C6H7O6Na) n , mainly composed of alginic acid (Alg);

[0045] (2) RGD series peptides,

[0046] (3) Polyglutamic acid (PGA),

[0047] (4) 0.1 M MES buffer (pH = 6.5),

[0048] (5)EDC(N-(3dimethylaminopropyl)-N'-ethyl carbodiimide hydrochloride),

[0049] (6)Sulfo-NHS (N-hydroxysulphosuccinimide),

[0050] (7) Hydroxylamine hydrochloride.

[0051] The specific steps for preparing the hydrogel material in this embodiment are as follows: using alginate as the skeleton material, activating the carboxyl groups distributed along the main chain of alginate, and then using amino reaction to modify the RGD series polypeptides and polyglutamic acid to the alginate skeleton material respectively to form a reprogrammed responsive intelligent hydrogel material, and adjusting the mechanical properties of the gel by regulating the viscosity (3-200mPa.s) and Ca2+ concentration.

[0052] (1) Obtaining Alg skeleton: Dissolve 1-20 mg / ml sodium alginate in deionized water

[0053] (2) Modification of the Alg skeleton with RGD series peptides:

[0054] The carboxyl groups distributed along the main chain of alginate are activated, and then the RGD series polypeptides (1-100 mM) are modified to the alginate backbone material by amino reaction.

[0055] (3) The Alg skeleton is modified with polyglutamic acid:

[0056] The carboxyl groups distributed along the main chain of alginate were activated, and then polyglutamic acid (1-100 mM) was modified to the side chain of the alginate skeleton material by amino reaction. The hydrogel prepared in this example was named 2.5-RL-PL. 2.5-RL-PL is the smart hydrogel material with the best effect.

[0057] In this example, the prepared reprogrammed responsive smart hydrogel material was characterized by optical microscopy and scanning electron microscopy. Figure 1 , Figure 1 A in the figure is the result of optical microscope observation. Figure 1 B scanning electron microscopy results.

[0058] This example also uses rheometer, nuclear magnetic resonance instrument and Fourier transform infrared spectrometer to characterize the prepared reprogrammed responsive smart hydrogel material. Figure 2 . Figure 2 A in FIG. 1 is the detection result of the elastic modulus of the reprogrammed responsive intelligent hydrogel material. Figure 2 B in the figure is the detection result of the elastic modulus of the reprogrammed responsive smart hydrogel material under different pH conditions. It can be seen that the reprogrammed responsive smart hydrogel material can respond to changes in pH, and its elastic modulus shows corresponding changes. Figure 2 C in the figure is the chemical component of the reprogrammed responsive intelligent hydrogel material detected by Fourier transform infrared spectrometer. It can be seen that the obtained hydrogel material has characteristic chemical bonds of RGD series polypeptides and polyglutamic acid.

[0059] Example 2 Hydrogel material mediates induction of somatic cells into iPS cells

[0060] This embodiment provides a method for obtaining induced pluripotent stem cells by using the hydrogel material prepared in Example 1 to mediate cell reprogramming.

[0061] 1. The reagents used in this embodiment are:

[0062] (1) Preparation of universal culture medium: high glucose DMEM with 10% fetal bovine serum, 100 U / ml penicillin and 100 U / ml streptomycin.

[0063] (2) Preparation of fibroblast culture medium: high-glucose DMEM supplemented with 10% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 100 U / ml penicillin, 100 U / ml streptomycin and 0.055 μM β-mercaptoethanol.

[0064] (3) Preparation of iPS culture medium: 10% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 0.055 μM β-mercaptoethanol, 100 U / ml penicillin, 100 U / ml streptomycin and 1000 U / ml LIF (Millipore) were added to high-glucose DMEM.

[0065] 2. The method provided in this embodiment for obtaining induced pluripotent stem cells by cell reprogramming mediated by hydrogel materials comprises the following specific steps:

[0066] (1) Lentiviral vector transfection of somatic cells

[0067] The virus was prepared using methods known in the art, with the pFUW-tetO vector as the backbone, and the pluripotency genes Oct4 (abbreviated as O), Sox2 (abbreviated as S), Klf4 (abbreviated as K), c-Myc (abbreviated as M) and rtTA (abbreviated as rtTA, a plasmid vector with a response element of doxycycline) were respectively constructed into lentiviral vectors. The above-mentioned lentiviral vector and packaging plasmid were introduced into HEK293T cells by liposome transfection. 24-96 hours after transfection, the virus supernatant was collected. Five viruses, O, S, K, M and rtTA, were mixed. The above-mentioned virus mixture was added to somatic cells, and fresh iPS culture medium was replaced after infection.

[0068] (2) Hydrogel-mediated cell reprogramming to obtain induced pluripotent stem cells

[0069] The infected somatic cells were resuspended in 1 ml of reprogrammed responsive smart hydrogel material, and the final concentration of somatic cells was 1×10 4 -300×10 4 , a hydrogel is formed at 37°C under the action of calcium ions, cultured for 7-10 days, and iPS cells are established.

[0070] Comparative Example 1 Induction effect of no hydrogel, conventional three-dimensional hydrogel material and reprogrammed responsive intelligent hydrogel material

[0071] This comparative example respectively performs cell reprogramming mediated by the classical method (i.e., without using a hydrogel material, denoted as TCP control); cell reprogramming mediated by a conventional three-dimensional hydrogel material without using an Alg skeleton modified by chemical molecules (denoted as 2.1 and 2.5, respectively); and compares the induction effect of cell reprogramming mediated by a reprogramming responsive intelligent hydrogel material (denoted as 2.5-RL-PL).

[0072] When the cell reprogramming reached the 10th day, alkaline phosphatase staining was performed on the samples of cell reprogramming mediated by the above different methods, and it was found that on the 10th day of cell reprogramming mediated by the classical method (i.e., without using hydrogel materials, denoted as TCP control), the cells positive for alkaline phosphatase staining were scattered and had not yet formed a typical morphology similar to embryonic stem cell clones; therefore, on the 10th day of cell reprogramming mediated by the classical method (i.e., without using hydrogel materials), the number of cell clones positive for alkaline phosphatase staining was 0.

[0073] The numbers of alkaline phosphatase-positive cell clones in cell reprogramming mediated by conventional three-dimensional hydrogel materials (respectively denoted as 2.1 and 2.5) and cell reprogramming mediated by reprogramming-responsive intelligent hydrogel materials (respectively denoted as 2.5-RL-PL) were counted, as shown in FIG. Figure 3As shown in B. The results show that compared with the classic reprogramming method, the reprogramming responsive smart hydrogel material can mediate cell reprogramming relatively faster; compared with cell reprogramming mediated by conventional three-dimensional hydrogel materials, the reprogramming responsive smart hydrogel material can mediate cell reprogramming more efficiently.

[0074] Comparative Example 2 Induction Effects Mediated by Different Pluripotency-Related Genes

[0075] This comparative example uses another set of pluripotency-related gene combinations (Oct4, Sox2, Klf4 and Glis1, referred to as OSKG) for cell reprogramming. Cell reprogramming mediated by the classical method (i.e., without using hydrogel materials, denoted as TCP control), cell reprogramming mediated by conventional three-dimensional hydrogel materials (denoted as 2.1 and 2.5, respectively), and cell reprogramming mediated by reprogramming responsive intelligent hydrogel materials (denoted as 2.5-RL-PL) were used respectively.

[0076] On the 10th day of reprogramming, alkaline phosphatase staining was performed on the cells reprogrammed by the above different methods, and then the number of cell clones with positive alkaline phosphatase staining was counted. Figure 3 C. The results show that compared with the classical reprogramming method, the reprogramming responsive intelligent hydrogel material can mediate cell reprogramming relatively faster; compared with cell reprogramming mediated by conventional three-dimensional hydrogel materials, the reprogramming responsive intelligent hydrogel material can mediate cell reprogramming more efficiently.

[0077] Experimental Example Identification of Induced Pluripotent Stem Cells

[0078] In this experimental example, the induced pluripotent stem cells obtained in the examples and comparative examples were tested.

[0079] 1. Statistical results of stem cell clone size

[0080] The ImagJ method was used to detect the clone size of the obtained stem cells.

[0081] Figure 3 A The results show that the statistical results show that compared with the traditional reprogramming method and the non-responsive hydrogel material, the reprogrammed responsive smart hydrogel material prepared in Example 1 is more conducive to the growth of cell clones. Among them, 2.1 represents a relatively hard conventional three-dimensional hydrogel material; 2.5 represents a relatively soft conventional three-dimensional hydrogel material; 2.5-RL-PL represents a reprogrammed responsive smart hydrogel material.

[0082] 2. Stem cell morphology identification and alkaline phosphatase staining identification

[0083] The induced pluripotent stem cells obtained in this example have a morphology similar to that of embryonic stem cells. Figure 4 As shown in A.

[0084] For alkaline phosphatase identification, the main reagents used are:

[0085] (1) Alkaline phosphatase staining kit

[0086] (2) Fixative

[0087] Alkaline phosphatase assay steps:

[0088] (1) Aspirate the cell culture medium and wash once with PBS.

[0089] (2) Use fixative and fix at room temperature for 10 minutes.

[0090] (3) Aspirate the fixative and wash once with TBST.

[0091] (4) Add 0.5 ml of alkaline phosphatase staining solution to each well of the 24-well plate and incubate at room temperature in the dark for about 20 min.

[0092] (5) Aspirate the alkaline phosphatase staining solution, wash once with PBS, and finally store in PBS solution.

[0093] Subsequently, alkaline phosphatase-positive clones can be observed under an optical microscope in the induced pluripotent stem cells obtained by reprogramming the responsive smart hydrogel material, such as Figure 4 As shown in B.

[0094] 3. Identification of the expression of pluripotency-related genes at the transcriptional level in pluripotent stem cells (iPS)

[0095] Identification of the expression of pluripotency-related genes at the transcriptional level in pluripotent stem cells (iPS) obtained by cell reprogramming mediated by reprogramming responsive intelligent hydrogel materials. RNA was extracted from induced pluripotent stem cells (iPS) obtained by reprogramming responsive intelligent hydrogel materials using the Trizol method. 0.5g RNA sample was taken for reverse transcription to generate cDNA, which was then detected and analyzed by quantitative PCR. The primer sequences used in the detection process are as follows:

[0096] (1) Esrrb:

[0097] Forward primer nucleotide sequence (SEQ ID NO.11): 5'-CACCTGCTAAAAAGCCATTGACT-3'

[0098] Reverse primer nucleotide sequence (SEQ ID NO.12): 5'-CAACCCCTAGTAGATTCGAGACGAT-3'

[0099] (2)UTF1:

[0100] Forward primer nucleotide sequence (SEQ ID NO.13): 5'-GGATGTCCCGGTGACTACGTCTG-3'

[0101] Reverse primer nucleotide sequence (SEQ ID NO.14): 5'-GGCGGATCTGGTTATCGAAGGGT-3'

[0102] (3)NANOG:

[0103] Forward primer nucleotide sequence (SEQ ID NO.15): 5'-TCTTCCTGGTCCCCACAGTTT-3'

[0104] Reverse primer nucleotide sequence (SEQ ID NO.16): 5'-GCAAGAATAGTTCTCGGGATGAA-3'

[0105] (4)OCT4:

[0106] Forward primer nucleotide sequence (SEQ ID NO.17): 5'-AGAGGATCACCTTGGGGTACA-3'

[0107] Reverse primer nucleotide sequence (SEQ ID NO.18): 5'-CGAAGCGACAGATGGTGGTC-3'

[0108] (5)Actin:

[0109] Forward primer nucleotide sequence (SEQ ID NO.19): 5'-GAGACCTTCAACACCCCAGC-3'

[0110] Reverse primer nucleotide sequence (SEQ ID NO.20): 5'-ATGTCACGCACGATTTCCC-3'

[0111] like Figure 5 As shown, the endogenous pluripotency-related genes Esrrb (A), Utf1 (B), Oct4 (C) and Nanog (D) in the pluripotent stem cells (iPS) induced by the reprogramming responsive smart hydrogel material showed significantly high expression.

[0112] 4. Identification of the expression of pluripotency-related genes at the translational level in pluripotent stem cells (iPS)

[0113] The expression of endogenous pluripotency-related genes OCT4 and SOX2 in induced pluripotent stem cells (iPS) obtained by reprogramming responsive smart hydrogel materials was detected by immunofluorescence staining. Figure 6 As shown, the endogenous pluripotency-related genes Oct4 and Sox2 were significantly overexpressed in induced pluripotent stem cells (iPS) formed by reprogramming responsive smart hydrogel materials.

[0114] 5. Identification of methylation patterns in specific regions of the promoters of pluripotency-related genes Oct4 and Nanog in pluripotency stem cells (iPS)

[0115] The samples of this example include MEF, pluripotent stem cells (iPS) (3D-resp-iPS-1# and 3D-resp-iPS-2#) obtained by cell reprogramming mediated by reprogramming responsive intelligent hydrogel materials, and embryonic stem cells (ESC). The primer information used in the specific experimental process is as follows:

[0116] (1)OCT4 promoter:

[0117] Forward primer (SEQ ID NO. 21): 5′-ATGGGTTGAAATATTGGGTTTATTTA-3′;

[0118] Reverse primer (SEQ ID NO. 22): 5'-CCACCCTCTAACCTTAACCTCTAAC-3'.

[0119] (2)NANOG promoter:

[0120] Forward primer (SEQ ID NO.23): 5'-GATTTTGTAGGTGGGATTAATTGTGAATTT-3;'

[0121] Reverse primer (SEQ ID NO. 24): 5'-ACCAAAAAAACCCACACTCATATCAATATA-3'.

[0122] like Figure 7 As shown, Figure 7 A is the methylation pattern of specific regions of Oct4 and Nanog promoters in MEFs; Figure 7 B is the methylation pattern of specific regions of Oct4 and Nanog promoters in embryonic stem cells (ESC); Figure 7C and D are the methylation patterns of specific regions of the Oct4 and Nanog promoters in the pluripotent stem cells (iPS) (respectively denoted as 3D-resp-iPS-1# and 3D-resp-iPS-2#) obtained by cell reprogramming mediated by the reprogramming responsive intelligent hydrogel material described in the present invention.

[0123] Figure 7 The results showed that the specific regions of Oct4 and Nanog promoters in the pluripotent stem cells (iPS) (respectively denoted as 3D-resp-iPS-1# and 3D-resp-iPS-2#) obtained by cell reprogramming mediated by the reprogramming responsive intelligent hydrogel material described in the present invention showed a complete demethylation pattern, and this pattern was similar to the methylation pattern of the specific regions of Oct4 and Nanog promoters in the positive control cells.

[0124] 6. Karyotype identification of pluripotent stem cells (iPS) obtained by cell reprogramming mediated by reprogramming responsive intelligent hydrogel materials

[0125] The karyotype of the pluripotent stem cells obtained in Example 2 was identified by using the method of treating with narcissin. The identification results are as follows: Figure 8 . Figure 8 The results showed that the iPS cells obtained in Example 2 had a normal karyotype.

[0126] The steps of karyotyping are as follows:

[0127] a. Ensure that the cells are in the logarithmic growth phase on the day of the experiment. Generally, one experiment can be performed in a 6 cm culture dish with a density of 80%.

[0128] b. Take the cells to be tested out of the 37°C, 5% CO2 incubator, add colchicine (final concentration 0.2ug / ml), shake well and incubate at 37°C for 1-2 hours.

[0129] c. While waiting, prepare a hypotonic solution of 0.075M KCL (preparation: 8ml sterile water + 44.7mg KCL) and preheat it in a 37℃ water bath.

[0130] d. After incubation, the cells were digested with trypsin, centrifuged at 1200 rpm for 5 min, collected in a centrifuge tube, the supernatant removed, resuspended in 8 ml of hypotonic solution, mixed well, and placed in a 37°C water bath for 40 min.

[0131] e. Add 2 ml of freshly prepared fixative (methanol / glacial acetic acid = 2 / 1 ~ 3 / 1) and mix well. After leaving at room temperature for 10 min, centrifuge at 1000 rpm for 10 min, remove the supernatant (leave 500 ul and blow the cells at the bottom evenly first), add 8 ml of new fixative, centrifuge at 1200 rpm for 10 min, repeat three times, drop on the slide, and age in an 80°C oven for 3 hours.

[0132] f. Place the dried slide in freshly prepared trypsin (0.25%) and digest for 1 minute (strictly control the time), then place it in Giemsa stain for 5-10 minutes (first stain 1 slide for 5 minutes, and adjust the subsequent staining time according to the color), take it out and rinse off the stain with running water, and observe the results under a microscope.

[0133] 7. Identification of in vivo differentiation ability

[0134] The iPS cells induced by the reprogrammed responsive intelligent hydrogel material obtained in Example 2 were injected subcutaneously into the thigh root of the recipient mouse, and the teratoma tissue was taken out 3-8 weeks after transplantation for hematoxylin-eosin staining. Fig. 9 As shown, the teratoma produced by pluripotent stem cells (iPS) obtained by cell reprogramming mediated by reprogramming responsive intelligent hydrogel materials contains tissue structures of three different germ layers, namely ectoderm, mesoderm and endoderm, proving the omnipotence of the iPS cell line.

[0135] 8. Identification of induction efficiency

[0136] The alkaline phosphatase staining method was used to detect the induction efficiency of pluripotent stem cells in Example 2 and Comparative Example 1. The results showed that in Example 2, the iPS cell induction efficiency was 512.3333 (+ / - 25.32456) for cell reprogramming mediated by the reprogramming-responsive intelligent hydrogel material-mediated stem cells (for every 10,000 starting cells), and the induction efficiency was about 5.12%.

[0137] Conventional three-dimensional hydrogel materials mediated cell reprogramming (for every 10,000 starting cells), and the number of alkaline phosphatase-positive cell clones obtained was 238.6667 (+ / -9.291573), with an induction efficiency of about 2.38%.

[0138] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention. Sequence Listing <110> National Center for Nanoscience and Technology <120> A reprogrammed responsive intelligent hydrogel material and its preparation method and application <130> KHP221111603.7 <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> 1059 <212> DNA <213> Artificial Sequence <400> 1 atggctggac acctggcttc agacttcgcc ttctcacccc caccaggtgg gggtgatggg 60 tcagcagggc tggagccggg ctgggtggat cctcgaacct ggctaagctt ccaagggcct 120 ccaggtgggc ctggaatcgg accaggctca gaggtattgg ggatctcccc atgtccgccc 180 gcatacgagt tctgcggagg gatggcatac tgtggacctc aggttggact gggcctagtc 240 ccccaagttg gcgtggagac tttgcagcct gagggccagg caggagcacg agtggaaagc 300 aactcagagg gaacctcctc tgagccctgt gccgaccgcc ccaatgccgt gaagttggag 360 aaggtggaac caactcccga ggagtcccag gacatgaaag ccctgcagaa ggagctagaa 420 cagtttgcca agctgctgaa gcagaagagg atcaccttgg ggtacaccca ggccgacgtg 480 gggctcaccc tgggcgttct ctttggaaag gtgttcagcc agaccaccat ctgtcgcttc 540 gggctcaccc tgggcgttct ctttggaaag gtgttcagcc agaccaccat ctgtcgcttc 540 gaggccttgc agctcagcct taagaacatg tgtaagctgc ggcccctgct ggagaagtgg 600 gaggccttgc agctcagcct taagaacatg tgtaagctgc ggcccctgct ggagaagtgg 600 gtggaggaag ccgacaacaa tgagaacctt caggagatat gcaaatcgga gaccctggtg 660 gtggaggaag ccgacaacaa tgagaacctt caggagatat gcaaatcgga gaccctggtg 660 caggcccgga agagaaagcg aactagcatt gagaaccgtg tgaggtggag tctggagacc 720 caggcccgga agagaaagcg aactagcatt gagaaccgtg tgaggtggag tctggagacc 720 atgtttctga agtgcccgaa gccctcccta cagcagatca ctcacatcgc caatcagctt 780 atgtttctga agtgcccgaa gccctcccta cagcagatca ctcacatcgc caatcagctt 780 gggctagaga aggatgtggt tcgagtatgg ttctgtaacc ggcgccagaa gggcaaaaga 840 gggctagaga aggatgtggt tcgagtatgg ttctgtaacc ggcgccagaa gggcaaaaga 840 tcaagtattg agtattccca acgagaagag tatgaggcta cagggacacc tttcccaggg 900 tcaagtattg agtattccca acgagaagag tatgaggcta cagggacacc tttcccaggg 900 ggggctgtat cctttcctct gcccccaggt ccccactttg gcaccccagg ctatggaagc 960 ggggctgtat cctttcctct gcccccaggt ccccactttg gcaccccagg ctatggaagc 960 ccccacttca ccacactcta ctcagtccct tttcctgagg gcgaggcctt tccctctgtt 1020 ccccacttca ccacactcta ctcagtccct tttcctgagg gcgaggcctt tccctctgtt 1020 cccgtcactg ctctgggctc tcccatgcat tcaaactga 1059 cccgtcactg ctctgggctc tcccatgcat tcaaactga 1059 <210> 2<210> 2 <211> 352 <211> 352 <212> PRT <212> PRT <213> 人工序列(Artificial Sequence) <213> Artificial Sequence <400> 2 <400> 2 Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly Met Ala Gly His Leu Ala Ser Asp Phe Ala Phe Ser Pro Pro Pro Gly 1 5 10 15 Gly Gly Asp Gly Ser Ala Gly Leu Glu Pro Gly Trp Val Asp Pro Arg 20 25 30 Thr Trp Leu Ser Phe Gln Gly Pro Pro Gly Gly Pro Gly Ile Gly Pro 35 40 45 Gly Ser Glu Val Leu Gly Ile Ser Pro Cys Pro Pro Ala Tyr Glu Phe 50 55 60 Cys Gly Gly Met Ala Tyr Cys Gly Pro Gln Val Gly Leu Gly Leu Val 65 70 75 80 Pro Gln Val Gly Val Glu Thr Leu Gln Pro Glu Gly Gln Ala Gly Ala 85 90 95 Arg Val Glu Ser Asn Ser Glu Gly Thr Ser Ser Glu Pro Cys Ala Asp 100 105 110 Arg Pro Asn Ala Val Lys Leu Glu Lys Val Glu Pro Thr Pro Glu Glu 115 120 125 Ser Gln Asp Met Lys Ala Leu Gln Lys Glu Leu Glu Gln Phe Ala Lys 130 135 140 Leu Leu Lys Gln Lys Arg Ile Thr Leu Gly Tyr Thr Gln Ala Asp Val 145 150 155 160 Gly Leu Thr Leu Gly Val Leu Phe Gly Lys Val Phe Ser Gln Thr Thr 165 170 175 Ile Cys Arg Phe Glu Ala Leu Gln Leu Ser Leu Lys Asn Met Cys Lys 180 185 190 Leu Arg Pro Leu Leu Glu Lys Trp Val Glu Glu Ala Asp Asn Asn Glu 195 200 205 Asn Leu Gln Glu Ile Cys Lys Ser Glu Thr Leu Val Gln Ala Arg Lys 210 215 220 Arg Lys Arg Thr Ser Ile Glu Asn Arg Val Arg Trp Ser Leu Glu Thr 225 230 235 240 Met Phe Leu Lys Cys Pro Lys Pro Ser Leu Gln Gln Ile Thr His Ile 245 250 255 Ala Asn Gln Leu Gly Leu Glu Lys Asp Val Val Arg Val Trp Phe Cys 260 265 270 Asn Arg Arg Gln Lys Gly Lys Arg Ser Ser Ile Glu Tyr Ser Gln Arg 275 280 285 Glu Glu Tyr Glu Ala Thr Gly Thr Pro Phe Pro Gly Gly Ala Val Ser 290 295 300 Phe Pro Leu Pro Pro Gly Pro His Phe Gly Thr Pro Gly Tyr Gly Ser 305 310 315 320 Pro His Phe Thr Thr Leu Tyr Ser Val Pro Phe Pro Glu Gly Glu Ala 325 330 335 Phe Pro Ser Val Pro Val Thr Ala Leu Gly Ser Pro Met His Ser Asn 340 345 350 <210> 3 <211> 1452 <212> DNA <213> Artificial Sequence <400> 3 atgaggcagc cacctggcga gtctgacatg gctgtcagcg acgctctgct cccgtccttc 60 tccacgttcg cgtccggccc ggcgggaagg gagaagacac tgcgtccagc aggtgccccg 120 actaaccgtt ggcgtgagga actctctcac atgaagcgac ttcccccact tcccggccgc 180 ccctacgacc tggcggcgac ggtggccaca gacctggaga gtggcggagc tggtgcagct 240 tgcagcagta acaacccggc cctcctagcc cggagggaga ccgaggagtt caacgacctc 300 ctggacctag actttatcct ttccaactcg ctaacccacc aggaatcggt ggccgccacc 360 gtgaccacct cggcgtcagc ttcatcctcg tcttccccgg cgagcagcgg ccctgccagc 420 gcgccctcca cctgcagctt cagctatccg atccgggccg ggggtgaccc gggcgtggct 480 gccagcaaca caggtggagg gctcctctac agccgagaat ctgcgccacc tcccacggcc 540 cccttcacc tggcggacat caatgacgtg agcccctcgg gcggctcgt ggctgagctc 600 ctgcggccgg agttggaccc agtatacatt ccgccacagc agcctcagcc gccaggtggc 660 gggctgatgg gcaagtttgt gctgaaggcg tctctgacca cccctggcag cgagtacagc 720 agcccttcgg tcatcagtgt tagcaagga agcccagacg gcagccaccc cgtggtagtg 780 gcgccctaca gcggtggccc gccgcgcatg tgcccaga ttaagcaaga ggcggtcccg 840 tcctgcacgg tcagccggtc cctagaggcc cattgagcg ctggacccca gctcagcac 900 ggccaccggc cccacaca cgactcccc ctggggcggc agctccccac caggactacc 960 cctacactga gtcccgagga actgctgaac agcagggact gtcaccctgg cctgcctt 1020 cccccaggat tccatcccca tccggggccc aactaccctc ctttcctgcc agaccagatg 1080 cagtcacaag tcccctctct ccattatcaa gagctcatgc caccggttc ctgcctgcca 1140 gaggagcccca agccaaagg gggaagaagg tcgtggcccc ggaaaagaac agccaccac 1200 acttgtgact atgcaggctg tgcaaaacc tataccaga gttctcatct caggcacac 1260 ctgcgaactc acacaggcga gaaaccttac cactgtgact gggacggctg tgggtggaaa 1320 ttcgcccgct ccgatgaact gaccaggcac taccgcaaac acacagggca ccggcccttt 1380 cagtgccaga agtgtgacag ggccttttcc aggtcggacc accttgcctt acacatgaag 1440 aggcactttt aa 1452 <210> 4 <211> 483 <212> PRT <213> Artificial Sequence <400> 4 Met Arg Gln Pro Pro Gly Glu Ser Asp Met Ala Val Ser Asp Ala Leu 1 5 10 15 Leu Pro Ser Phe Ser Thr Phe Ala Ser Gly Pro Ala Gly Arg Glu Lys 20 25 30 Thr Leu Arg Pro Ala Gly Ala Pro Thr Asn Arg Trp Arg Glu Glu Leu 35 40 45 Ser His Met Lys Arg Leu Pro Pro Leu Pro Gly Arg Pro Tyr Asp Leu 50 55 60 Ala Ala Thr Val Ala Thr Asp Leu Glu Ser Gly Gly Ala Gly Ala Ala 65 70 75 80 Cys Ser Ser Asn Asn Pro Ala Leu Leu Ala Arg Arg Glu Thr Glu Glu 85 90 95 Phe Asn Asp Leu Leu Asp Leu Asp Phe Ile Leu Ser Asn Ser Leu Thr 100 105 110 His Gln Glu Ser Val Ala Ala Thr Val Thr Thr Ser Ala Ser Ala Ser 115 120 125 Ser Ser Ser Ser Pro Ala Ser Ser Gly Pro Ala Ser Ala Pro Ser Thr 130 135 140 Cys Ser Phe Ser Tyr Pro Ile Arg Ala Gly Gly Asp Pro Gly Val Ala 145 150 155 160 Ala Ser Asn Thr Gly Gly Gly Leu Leu Tyr Ser Arg Glu Ser Ala Pro 165 170 175 Pro Pro Thr Ala Pro Phe Asn Leu Ala Asp Ile Asn Asp Val Ser Pro 180 185 190 Ser Gly Gly Phe Val Ala Glu Leu Leu Arg Pro Glu Leu Asp Pro Val 195 200 205 Tyr Ile Pro Pro Gln Gln Pro Gln Pro Pro Gly Gly Gly Leu Met Gly 210 215 220 Lys Phe Val Leu Lys Ala Ser Leu Thr Thr Pro Gly Ser Glu Tyr Ser 225 230 235 240 Ser Pro Ser Val Ile Ser Val Ser Lys Gly Ser Pro Asp Gly Ser His 245 250 255 Pro Val Val Val Ala Pro Tyr Ser Gly Gly Pro Pro Arg Met Cys Pro 260 265 270 Lys Ile Lys Gln Glu Ala Val Pro Ser Cys Thr Val Ser Arg Ser Leu 275 280 285 Glu Ala His Leu Ser Ala Gly Pro Gln Leu Ser Asn Gly His Arg Pro 290 295 300 Asn Thr His Asp Phe Pro Leu Gly Arg Gln Leu Pro Thr Arg Thr Thr 305 310 315 320 Pro Thr Leu Ser Pro Glu Glu Leu Leu Asn Ser Arg Asp Cys His Pro 325 330 335 Gly Leu Pro Leu Pro Pro Gly Phe His Pro His Pro Gly Pro Asn Tyr 340 345 350 Pro Pro Phe Leu Pro Asp Gln Met Gln Ser Gln Val Pro Ser Leu His 355 360 365 Tyr Gln Glu Leu Met Pro Pro Gly Ser Cys Leu Pro Glu Glu Pro Lys 370 375 380 Pro Lys Arg Gly Arg Arg Ser Trp Pro Arg Lys Arg Thr Ala Thr His 385 390 395 400 Thr Cys Asp Tyr Ala Gly Cys Gly Lys Thr Tyr Thr Lys Ser Ser His 405 410 415 Leu Lys Ala His Leu Arg Thr His Thr Gly Glu Lys Pro Tyr His Cys 420 425 430 Asp Trp Asp Gly Cys Gly Trp Lys Phe Ala Arg Ser Asp Glu Leu Thr 435 440 445 Arg His Tyr Arg Lys His Thr Gly His Arg Pro Phe Gln Cys Gln Lys 450 455 460 Cys Asp Arg Ala Phe Ser Arg Ser Asp His Leu Ala Leu His Met Lys 465 470 475 480 Arg His Phe <210> 5 <211> 960 <212> DNA <213> Artificial Sequence <400> 5 atgtataaca tgatggagac ggagctgaag ccgccgggcc cgcagcaagc ttcggggggc 60 ggcggcggag gaggcaacgc cacggcggcg gcgaccggcg gcaaccagaa gaacagcccg 120 gaccgcgtca agaggcccat gaacgccttc atggtatggt cccgggggca gcggcgtaag 180 atggcccagg agaaccccaa gatgcacaac tcggagatca gcaagcgcct gggcgcggag 240 tggaaacttt tgtccgagac cgagaagcgg ccgttcatcg acgaggccaa gcggctgcgc 300 gctctgcaca tgaaggagca cccggattat aaataccggc cgcggcgga aaccaagacg ctcatgaaga aggataagta cacgcttccc ggaggcttgc tggcccccgg cgggaacagc 420 atggcgagcg gggttggggt gggcgccggc ctgggtgcgg gcgtgaacca gcgcatggac 480 agctacgcgc acatgaacgg ctggagcaac ggcagctaca gcatgatgca ggagcagctg ggctacccgc agcaccggg cctcaacgct cacggcgcgg cacagatgca accgatgcac cgctacgacg tcagcgccct gcagtacaac tccatgacca gctcgcagac ctacatgaac ggctcgccca cctacagcat gtcctactcg cagcagggca cccccggtat ggcgctgggc 720 tccatgggct ctgtggtcaa gtccgaggcc agctccagcc cccccgtggt tacctcttcc 780 tcccactcca gggcgccctg ccaggccggg gacctccggg acatgatcag catgtacctc 840 cccggcgccg aggtgccgga gcccgctgcg cccagtagac tgcacatggc ccagcactac cagagcggcc cggtgcccgg cacggccatt aacggcacac tgcccctgtc gcacatgtga 960 <210> 6 <211> 319 <212> PRT <213> Artificial Sequence <400> 6 Met Tyr Asn Met Met Glu Thr Glu Leu Lys Pro Pro Gly Pro Gln Gln 1 5 10 15 Ala Ser Gly Gly Gly Gly Gly Gly Gly Asn Ala Thr Ala Ala Ala Thr 20 25 30 Gly Gly Asn Gln Lys Asn Ser Pro Asp Arg Val Lys Arg Pro Met Asn 35 40 45 Ala Phe Met Val Trp Ser Arg Gly Gln Arg Arg Lys Met Ala Gln Glu 50 55 60 Asn Pro Lys Met His Asn Ser Glu Ile Ser Lys Arg Leu Gly Ala Glu 65 70 75 80 Trp Lys Leu Leu Ser Glu Thr Glu Lys Arg Pro Phe Ile Asp Glu Ala 85 90 95 Lys Arg Leu Arg Ala Leu His Met Lys Glu His Pro Asp Tyr Lys Tyr 100 105 110 Arg Pro Arg Arg Lys Thr Lys Thr Leu Met Lys Lys Asp Lys Tyr Thr 115 120 125 Leu Pro Gly Gly Leu Leu Ala Pro Gly Gly Asn Ser Met Ala Ser Gly 130 135 140 Val Gly Val Gly Ala Gly Leu Gly Ala Gly Val Asn Gln Arg Met Asp 145 150 155 160 Ser Tyr Ala His Met Asn Gly Trp Ser Asn Gly Ser Tyr Ser Met Met 165 170 175 Gln Glu Gln Leu Gly Tyr Pro Gln His Pro Gly Leu Asn Ala His Gly 180 185 190 Ala Ala Gln Met Gln Pro Met His Arg Tyr Asp Val Ser Ala Leu Gln 195 200 205 Tyr Asn Ser Met Thr Ser Ser Gln Thr Tyr Met Asn Gly Ser Pro Thr 210 215 220 Tyr Ser Met Ser Tyr Ser Gln Gln Gly Thr Pro Gly Met Ala Leu Gly 225 230 235 240 Ser Met Gly Ser Val Val Lys Ser Glu Ala Ser Ser Ser Pro Pro Val 245 250 255 Val Thr Ser Ser Ser His Ser Arg Ala Pro Cys Gln Ala Gly Asp Leu 260 265 270 Arg Asp Met Ile Ser Met Tyr Leu Pro Gly Ala Glu Val Pro Glu Pro 275 280 285 Ala Ala Pro Ser Arg Leu His Met Ala Gln His Tyr Gln Ser Gly Pro 290 295 300 Val Pro Gly Thr Ala Ile Asn Gly Thr Leu Pro Leu Ser His Met 305 310 315 <210> 7 <211> 1365 <212> DNA <213> Artificial Sequence <400> 7 ctggatttcc tttgggcgtt ggaaaccccg cagacagcca cgacgatgcc cctcaacgtg 60 aacttcacca acaggaacta tgacctcgac tacgactccg tacagcccta tttcatctgc 120 gacgaggaag agaatttcta tcaccagcaa cagcagagcg agctgcagcc gcccgcgccc 180 agtgaggata tctggaagaa attcgagctg cttcccaccc cgcccctgtc cccgagccgc 240 cgctccgggc tctgctctcc atcctatgtt gcggtcgcta cgtccttctc cccaagggaa 300 gacgatgacg gcggcggtgg caacttctcc accgccgatc agctggagat gatgaccgag 360 ttacttggag gagacatggt gaaccagagc ttcatctgcg atcctgacga cgagaccttc 420 atcaagaaca tcatcatcca ggactgtatg tggagcggtt tctcagccgc tgccaagctg 480 gtctcggaga agctggcctc ctaccaggct gcgcgcaaag acagcaccag cctgagcccc 540 gcccgcgggc acagcgtctg ctccacctcc agcctgtacc tgcaggacct caccgccgcc 600 gcgtccgagt gcattgaccc ctcagtggtc tttccctacc cgctcaacga cagcagctcg 660 cccaaatcct gtacctcgtc cgattccacg gccttctctc cttcctcgga ctcgctgctg 720 tcctccgagt cctccccacg ggccagccct gagcccctag tgctgcatga ggagacaccg 780 cccaccacca gcagcgactc tgaagaagag caagaagatg aggaagaaat tgatgtggtg 840 tctgtggaga agaggcaaac ccctgccaag aggtcggagt cgggctcatc tccatcccga 900 ggccacagca aacctccgca cagcccactg gtcctcaaga ggtgccacgt ctccactcac 960 cagcacaact acgccgcacc cccctccaca aggaaggact atccagctgc caagagggcc 1020 aagttggaca gtggcagggt cctgaagcag atcagcaaca accgcaagtg ctccagcccc 1080 aggtcctcag acacggagga aaacgacaag aggcggacac acaacgtctt ggaacgtcag 1140 aggaggaacg agctgaagcg cagctttttt gccctgcgtg accagatccc tgaattggaa 1200 aacaacgaaa aggcccccaa ggtagtgatc ctcaaaaaag ccaccgccta catcctgtcc 1260 attcaagcag acgagcacaa gctcacctct gaaaaggact tattgaggaa acgacgagaa 1320 cagttgaaac acaaactcga acagcttcga aactctggtg cataa 1365 <210> 8 <211> 439 <212> PRT <213> Artificial Sequence <400> 8 Met Pro Leu Asn Val Asn Phe Thr Asn Arg Asn Tyr Asp Leu Asp Tyr 1 5 10 15 Asp Ser Val Gln Pro Tyr Phe Ile Cys Asp Glu Glu Glu Asn Phe Tyr 20 25 30 His Gln Gln Gln Gln Ser Glu Leu Gln Pro Pro Ala Pro Ser Glu Asp 35 40 45 Ile Trp Lys Lys Phe Glu Leu Leu Pro Thr Pro Pro Leu Ser Pro Ser 50 55 60 Arg Arg Ser Gly Leu Cys Ser Pro Ser Tyr Val Ala Val Ala Thr Ser 65 70 75 80 Phe Ser Pro Arg Glu Asp Asp Asp Gly Gly Gly Gly Asn Phe Ser Thr 85 90 95 Ala Asp Gln Leu Glu Met Met Thr Glu Leu Leu Gly Gly Asp Met Val 100 105 110 Asn Gln Ser Phe Ile Cys Asp Pro Asp Asp Glu Thr Phe Ile Lys Asn 115 120 125 Ile Ile Ile Gln Asp Cys Met Trp Ser Gly Phe Ser Ala Ala Ala Lys 130 135 140 Leu Val Ser Glu Lys Leu Ala Ser Tyr Gln Ala Ala Arg Lys Asp Ser 145 150 155 160 Thr Ser Leu Ser Pro Ala Arg Gly His Ser Val Cys Ser Thr Ser Ser 165 170 175 Leu Tyr Leu Gln Asp Leu Thr Ala Ala Ala Ser Glu Cys Ile Asp Pro 180 185 190 Ser Val Val Phe Pro Tyr Pro Leu Asn Asp Ser Ser Ser Pro Lys Ser 195 200 205 Cys Thr Ser Ser Asp Ser Thr Ala Phe Ser Pro Ser Ser Asp Ser Leu 210 215 220 Leu Ser Ser Glu Ser Ser Pro Arg Ala Ser Pro Glu Pro Leu Val Leu 225 230 235 240 His Glu Glu Thr Pro Pro Thr Thr Ser Ser Asp Ser Glu Glu Glu Gln 245 250 255 Glu Asp Glu Glu Glu Ile Asp Val Val Ser Val Glu Lys Arg Gln Thr 260 265 270 Pro Ala Lys Arg Ser Glu Ser Gly Ser Ser Pro Ser Arg Gly His Ser 275 280 285 Lys Pro Pro His Ser Pro Leu Val Leu Lys Arg Cys His Val Ser Thr 290 295 300 His Gln His Asn Tyr Ala Ala Pro Pro Ser Thr Arg Lys Asp Tyr Pro 305 310 315 320 Ala Ala Lys Arg Ala Lys Leu Asp Ser Gly Arg Val Leu Lys Gln Ile 325 330 335 Ser Asn Asn Arg Lys Cys Ser Ser Pro Arg Ser Ser Asp Thr Glu Glu 340 345 350 Asn Asp Lys Arg Arg Thr His Asn Val Leu Glu Arg Gln Arg Arg Asn 355 360 365 Glu Leu Lys Arg Ser Phe Phe Ala Leu Arg Asp Gln Ile Pro Glu Leu 370 375 380 Glu Asn Asn Glu Lys Ala Pro Lys Val Val Ile Leu Lys Lys Ala Thr 385 390 395 400 Ala Tyr Ile Leu Ser Ile Gln Ala Asp Glu His Lys Leu Thr Ser Glu 405 410 415 Lys Asp Leu Leu Arg Lys Arg Arg Glu Gln Leu Lys His Lys Leu Glu 420 425 430 Gln Leu Arg Asn Ser Gly Ala 435 <210> 9 <211> 2370 <212> DNA <213> Artificial Sequence <400> 9 atgcattgcg aggtggccga ggcactttcg gacaagaggc caaaggaggc ccctggtgct 60 cctggccagg gccgcgggcc tgtcagcctg ggagcgcaca tggccttcag gattgctgtg 120 agtggtggcg gctgcgggga cgggaacccg ctagacctgc tgcctcggct accggtgcca 180 ccaccacgtg cccacgatct ccttcggccc cggagccctc gagactatgg tgtgtccaag 240 accggcagcg ggaaggtgaa cgggagctac gggcacagct cagagaagag cctgctggac 300 ctggacctgg ccgagggtcc cagcccctcc tgccaccagg gtctgtttct tcctgcaggg 360 accccaccac cccggggtca cccccctgtc tgtgagaagc tgctgcactt cccccaccca 420 aacaggtcac ccagacctca ggctacgttt gtgaacggca gcctcccagc cgctcagcac 480 atcaagcaag aagccctacc ggactaccag gccatggtca gcgcccacac acccctgccc 540 acccactgcc gagccccatc gtccatgggt ctgccctcag acctggactt tccagaccga 600 ggcctcacca accctgcacc ttcctgctac cttctgggca atgaacccat ctcagacctg 660 ggtccccaac ccgaggccca cctccccgag ggcagcctga aacgctgctg cctcctgggc 720 ctgcccccca cctcttcagc ctcctcctca ccctgtgcct cctcagatat caatcctgtc 780 atccactcct cccagacagc tctagttagc tgtgtaaatg gactccgaag cccacctctg 840 ccgggagacc tggggggccc tcccaagcgg tcacggcccg ggcctgcatc cagtgacggc 900 caggagggca gcttgcagct tgaagcatgc cggaagtcag gcttcctgaa gcaggagccc 960 atggacgagt tttcagagct ttttgctcca caccaccagg gtttgccacc cccttacccc 1020 ttgcctcagt tgccaactgg ccccggcctc ggaggcctag ggctgggcct ggcaggtagg 1080 atggttgccg gtcggcaggc atgccgctgg gtggactgct gcgcagccta cgagcagcag 1140 gaggagctgg tgcggcacat cgagaagagc cacatcgacc agcgcaaggg cgaagacttc 1200 acctgcttct gggccgggtg tgtgcggcgc tacaagccct tcaatgcccg ctacaagctg 1260 ctcatccaca tgagggtaca ctcaggcgag aagcccaaca agtgcatgtt cgaaggctgc 1320 agtaaagcct tttcccgtct ggagaacctg aagatccatc tgcggagcca cacaggcgag 1380 aaaccatacc tgtgccagca cccaggctgc cagaaggcct tcagcaactc cagcgaccgt 1440 gccaagcacc aacgcaccca cctcgacacg aagccatatg cttgtcagat ccctggctgc 1500 tccaagcgct acacggaccc cagctccctc cgcaagcacg tgaaggccca ctcagccaaa 1560 gagcagcagg tgcgtaagaa gctgcacaca ggtgccgacc cagaggctga tgttctgtcc 1620 gagtgtctgt ccctgcagca gctccaagca tccacactgt tgccggccag cagagggaag 1680 ggcagccaaa ccctgagcca ggagctcctc ccaggtgtgt atcctggctc cgtcacccca 1740 caaaacgggc ttgcttcagg catcctgtcc ccctcccacg atgtcccttc caggcaccac 1800 ccactggagg tccccactgg ttcccaccac cacctgtccc ctctgcccac agctgagagc 1860 accagggatg gcctggggcc cagtctcctt tcacccatgg tcagcccact gaaggggctt 1920 ggtcccccac cgctaccacc agcctcccag agtcagtctc cagggggaca gtcattctct 1980 acagtcccca gcaagcctac ctacccatcc ttccaaagcc caccacctct gcccagcccc 2040 caaggctacc aaggcagttt ccattccatc cagaactgct tcccctacgc tgactgctac 2100 cgggccactg agccagcagc ctccagggat ggactggtgg gtgatgccca cggtttcaac 2160 cccttgcgac ccagcacata ctccagcctc agcacacctt tatccgcacc aggctacgag 2220 accctggcag aaacgccgtg tcccccagcg ctgcagccac agccagctga agacctggta 2280 cctagtggtc ctgaggactg tggcttcttc cccaatgggg cctttgacca ctgtctgagt 2340 cacatcccgt ccatctacac tgacacctga 2370 <210> 10 <211> 789 <212> PRT <213> Artificial Sequence <400> 10 Met His Cys Glu Val Ala Glu Ala Leu Ser Asp Lys Arg Pro Lys Glu 1 5 10 15 Ala Pro Gly Ala Pro Gly Gln Gly Arg Gly Pro Val Ser Leu Gly Ala 20 25 30 His Met Ala Phe Arg Ile Ala Val Ser Gly Gly Gly Cys Gly Asp Gly 35 40 45 Asn Pro Leu Asp Leu Leu Pro Arg Leu Pro Val Pro Pro Pro Arg Ala 50 55 60 His Asp Leu Leu Arg Pro Arg Ser Pro Arg Asp Tyr Gly Val Ser Lys 65 70 75 80 Thr Gly Ser Gly Lys Val Asn Gly Ser Tyr Gly His Ser Ser Glu Lys 85 90 95 Ser Leu Leu Asp Leu Asp Leu Ala Glu Gly Pro Ser Pro Ser Cys His 100 105 110 Gln Gly Leu Phe Leu Pro Ala Gly Thr Pro Pro Pro Arg Gly His Pro 115 120 125 Pro Val Cys Glu Lys Leu Leu His Phe Pro His Pro Asn Arg Ser Pro 130 135 140 Arg Pro Gln Ala Thr Phe Val Asn Gly Ser Leu Pro Ala Ala Gln His 145 150 155 160 Ile Lys Gln Glu Ala Leu Pro Asp Tyr Gln Ala Met Val Ser Ala His 165 170 175 Thr Pro Leu Pro Thr His Cys Arg Ala Pro Ser Ser Met Gly Leu Pro 180 185 190 Ser Asp Leu Asp Phe Pro Asp Arg Gly Leu Thr Asn Pro Ala Pro Ser 195 200 205 Cys Tyr Leu Leu Gly Asn Glu Pro Ile Ser Asp Leu Gly Pro Gln Pro 210 215 220 Glu Ala His Leu Pro Glu Gly Ser Leu Lys Arg Cys Cys Leu Leu Gly 225 230 235 240 Leu Pro Pro Thr Ser Ser Ala Ser Ser Ser Pro Cys Ala Ser Ser Asp 245 250 255 Ile Asn Pro Val Ile His Ser Ser Gln Thr Ala Leu Val Ser Cys Val 260 265 270 Asn Gly Leu Arg Ser Pro Pro Leu Pro Gly Asp Leu Gly Gly Pro Pro 275 280 285 Lys Arg Ser Arg Pro Gly Pro Ala Ser Ser Asp Gly Gln Glu Gly Ser 290 295 300 Leu Gln Leu Glu Ala Cys Arg Lys Ser Gly Phe Leu Lys Gln Glu Pro 305 310 315 320 Met Asp Glu Phe Ser Glu Leu Phe Ala Pro His His Gln Gly Leu Pro 325 330 335 Pro Pro Tyr Pro Leu Pro Gln Leu Pro Thr Gly Pro Gly Leu Gly Gly 340 345 350 Leu Gly Leu Gly Leu Ala Gly Arg Met Val Ala Gly Arg Gln Ala Cys 355 360 365 Arg Trp Val Asp Cys Cys Ala Ala Tyr Glu Gln Gln Glu Glu Leu Val 370 375 380 Arg His Ile Glu Lys Ser His Ile Asp Gln Arg Lys Gly Glu Asp Phe 385 390 395 400 Thr Cys Phe Trp Ala Gly Cys Val Arg Arg Tyr Lys Pro Phe Asn Ala 405 410 415 Arg Tyr Lys Leu Leu Ile His Met Arg Val His Ser Gly Glu Lys Pro 420 425 430 Asn Lys Cys Met Phe Glu Gly Cys Ser Lys Ala Phe Ser Arg Leu Glu 435 440 445 Asn Leu Lys Ile His Leu Arg Ser His Thr Gly Glu Lys Pro Tyr Leu 450 455 460 Cys Gln His Pro Gly Cys Gln Lys Ala Phe Ser Asn Ser Ser Asp Arg 465 470 475 480 Ala Lys His Gln Arg Thr His Leu Asp Thr Lys Pro Tyr Ala Cys Gln 485 490 495 Ile Pro Gly Cys Ser Lys Arg Tyr Thr Asp Pro Ser Ser Leu Arg Lys 500 505 510 His Val Lys Ala His Ser Ala Lys Glu Gln Gln Val Arg Lys Lys Leu 515 520 525 His Thr Gly Ala Asp Pro Glu Ala Asp Val Leu Ser Glu Cys Leu Ser 530 535 540 Leu Gln Gln Leu Gln Ala Ser Thr Leu Leu Pro Ala Ser Arg Gly Lys 545 550 555 560 Gly Ser Gln Thr Leu Ser Gln Glu Leu Leu Pro Gly Val Tyr Pro Gly 565 570 575 Ser Val Thr Pro Gln Asn Gly Leu Ala Ser Gly Ile Leu Ser Pro Ser 580 585 590 His Asp Val Pro Ser Arg His His Pro Leu Glu Val Pro Thr Gly Ser 595 600 605 His His His Leu Ser Pro Leu Pro Thr Ala Glu Ser Thr Arg Asp Gly 610 615 620 Leu Gly Pro Ser Leu Leu Ser Pro Met Val Ser Pro Leu Lys Gly Leu 625 630 635 640 Gly Pro Pro Pro Leu Pro Pro Ala Ser Gln Ser Gln Ser Pro Gly Gly 645 650 655 Gln Ser Phe Ser Thr Val Pro Ser Lys Pro Thr Tyr Pro Ser Phe Gln 660 665 670 Ser Pro Pro Pro Leu Pro Ser Pro Gln Gly Tyr Gln Gly Ser Phe His 675 680 685 Ser Ile Gln Asn Cys Phe Pro Tyr Ala Asp Cys Tyr Arg Ala Thr Glu 690 695 700 Pro Ala Ala Ser Arg Asp Gly Leu Val Gly Asp Ala His Gly Phe Asn 705 710 715 720 Pro Leu Arg Pro Ser Thr Tyr Ser Ser Leu Ser Thr Pro Leu Ser Ala 725 730 735 Pro Gly Tyr Glu Thr Leu Ala Glu Thr Pro Cys Pro Pro Ala Leu Gln 740 745 750 Pro Gln Pro Ala Glu Asp Leu Val Pro Ser Gly Pro Glu Asp Cys Gly 755 760 765 Phe Phe Pro Asn Gly Ala Phe Asp His Cys Leu Ser His Ile Pro Ser 770 775 780 Ile Tyr Thr Asp Thr 785 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <400> 11 cacctgctaa aaagccattg act 23 <210> 12 <211> 25 <212> DNA <213> Artificial Sequence <400> 12 caacccctag tagattcgag acgat 25 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <400> 13 ggatgtcccg gtgactacgt ctg 23 <210> 14 <211> twenty three <212> DNA <213> Artificial Sequence <400> 14 ggcggatctg gttatcgaag ggt 23 <210> 15 <211> twenty one <212> DNA <213> Artificial Sequence <400> 15 tcttcctggt ccccacagtt t 21 <210> 16 <211> twenty three <212> DNA <213> Artificial Sequence <400> 16 gcaagaatag ttctcgggat gaa 23 <210> 17 <211> twenty one <212> DNA <213> Artificial Sequence <400> 17 agaggatcac cttggggtac a 21 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <400> 18 cgaagcgaca gatggtggtc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <400> 19 gagaccttca acaccccagc 20 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <400> 20 atgtcacgca cgatttccc 19 <210> twenty one <211> 26 <212> DNA <213> Artificial Sequence <400> twenty one atgggttgaa atattgggtttattta 26 <210> twenty two <211> 25 <212> DNA <213> Artificial Sequence <400> twenty two cccaccctcta accttaacct ctaac 25 <210> twenty three <211> 30 <212> DNA <213> Artificial Sequence <400> twenty three gattttgtag gtgggattaa ttgtgaattt 30 <210> twenty four <211> 30 <212> DNA <213> Artificial Sequence <400> twenty four accaaaaaaa cccacactca tatcaatata 30

Claims

1. A reprogrammable responsive smart hydrogel material, characterized in that: The hydrogel material uses alginate as a skeleton, and chemical molecules are used to modify the skeleton, wherein the chemical molecules are RGD series polypeptides and polyglutamic acid.

2. The method for preparing the reprogrammed responsive smart hydrogel material according to claim 1, characterized in that: include: Using alginate as the skeleton material, the side chain of alginate is subjected to a carboxyl activation reaction, and the RGD series polypeptides and polyglutamic acid are modified to the side chains of the alginate skeleton material respectively by amino reaction to form a reprogrammed responsive intelligent hydrogel material.

3. Use of the reprogramming-responsive intelligent hydrogel material according to claim 1 in mediating somatic cell reprogramming to obtain induced pluripotent stem cells.

4. A method for obtaining induced pluripotent stem cells by reprogramming somatic cells, characterized in that: The reprogramming responsive smart hydrogel material according to claim 1 is used as an induction medium.

5. The method according to claim 4, characterized in that Somatic cell reprogramming is mediated by proteins encoded by pluripotency-associated genes.

6. The method according to claim 5, characterized in that The pluripotency-related genes include but are not limited to SOX2, Sall4, OCT4, Zfp42, Nr5a2, Daxl, Eras, Esrrb, Dppa2, Lin28A, Utf1, Klf4, Glis1, Prdm1, Prdm14, c-myc, and Tbx5.

7. The method according to claim 6, characterized in that The pluripotency-related gene is selected from one of Sox2, Oct4, c-myc and Klf4.

8. The method according to claim 6, characterized in that The nucleotide sequences of the pluripotency-related genes are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 and SEQ ID NO.

9.

9. The method according to claim 5, characterized in that The protein is one or more of the proteins encoded by Sox2, Oct4, c-myc and Klf4 genes of mice, rats, pigs, cattle, cynomolgus monkeys or humans.

10. The method according to claim 9, characterized in that The amino acid sequence of the protein is shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, SEQ ID NO.8 and / or SEQ ID NO.

10.

11. The method according to claim 4, characterized in that The somatic cells are somatic cells derived from mammals.

12. The method according to claim 11, characterized in that The somatic cells include blood cells, skin cells, Sertoli cells and / or urine epithelial cells.

13. The method according to any one of claims 4 to 12, characterized in that: include: (1) using one or more of the pluripotency-related genes SOX2, Sall4, OCT4, Zfp42, Nr5a2, Daxl, Eras, Esrrb, Dppa2, Lin28A, Utf1, Klf4, Glis1, Prdm1, Prdm14, c-myc, and Tbx5 to construct expression vectors and treat somatic cells; The treatment is to introduce the nucleotide sequence, amino acid sequence or expression vector of the pluripotency-related gene into the somatic cell; (2) The treated somatic cells obtained in step (1) are inoculated into a reprogramming responsive smart hydrogel material to screen and obtain induced pluripotent stem cells.

14. The method according to claim 13, characterized in that The treatment includes plasmid transfection, electrofection, virus infection, mRNA transfection and / or protein transduction.

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