Myocardial cell reprogramming method, related preparation and application

Abstract

The invention provides a myocardial cell reprogramming method, a related preparation and application. The invention provides a myocardial cell reprogramming method which comprises the following steps: carrying out balling culture on myofibroblasts on bionic interpenetrating network hydrogel, so that the myofibroblasts are gathered, and reprogramming the myofibroblasts into myocardial-like cells. The myofibroblasts used before culture are in an activated state. According to the method for realizing cell reprogramming based on the bionic interpenetrating network hydrogel, cell nucleus transplantation, virus transfection or gene editing are not needed, additional chemical induction reagents are not needed, the safety is high, and the effect is remarkable. And culturing supernate based on the reprogrammed cell balls for treating myocardial fibrosis.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a method for reprogramming cardiomyocytes and related preparations and applications. Background Art

[0002] Due to the complex cardiovascular structure and the limited regenerative capacity of adult cardiomyocytes, cardiovascular disease (CVD) has diverse etiologies and is difficult to cure, remaining a leading cause of death in humans. Studies have confirmed that the ultimate pathology of nearly all cardiovascular diseases, including hypertension, hypertrophic cardiomyopathy, arrhythmias, and myocardial infarction, is pathological myocardial fibrosis, which in turn increases the risk of heart failure and sudden cardiac death. Furthermore, many cardiovascular diseases are associated with aging, and the frequent presence of myocardial fibrosis in these diseases suggests a close relationship between aging and myocardial fibrosis. Myocardial fibrosis, which develops during aging, is a key characteristic of cardiac aging and can lead to structural disorder, increased stiffness, and decreased cardiac compliance and diastolic function. Therefore, reversing or ameliorating myocardial fibrosis is of great significance for the treatment of various cardiovascular diseases and cardiac aging. However, current clinical interventions and treatments for myocardial fibrosis remain very limited.

[0003] Based on existing research evidence, specific cytokines, growth factors, matrix cell proteins, integrins, and mechanically sensitive signaling pathways have a significant impact on the differentiation of cardiac fibroblasts into myofibroblasts. Therefore, a number of anti-fibrotic drug treatment strategies based on blocking or inhibiting these factors have been proposed. In addition to drug therapy, cell-based therapies are also considered to be potential treatments for reversing myocardial fibrosis, including stem cell therapy, cardiomyocyte transplantation, gene therapy, and immunotherapy. However, whether it is drug therapy or cell therapy, the safety and efficacy of clinical treatment still need to be further confirmed, and the complexity of cell therapy itself also limits the promotion of its clinical application. There is an urgent need for safe and effective methods to reverse myocardial cell fibrosis. Summary of the Invention

[0004] The purpose of this application is to provide a method for reprogramming myocardial cells and related preparations and applications to improve myocardial cell fibrosis.

[0005] In order to achieve the purpose of the present application, in a first aspect, the present application provides a method for reprogramming cardiomyocytes, comprising culturing myofibroblasts into spheres on a biomimetic interpenetrating network hydrogel so that the myofibroblasts aggregate and reprogram the myofibroblasts into cardiomyocyte-like cells.

[0006] Preferably, the myofibroblasts used before culture are in an activated state. In this application, the activated state refers to the primary cardiac fibroblasts expressing a large amount of proteins such as α-SMA, Collagen I and Collagen III after being cultured with TCP, and the cardiac fibroblasts are activated into myofibroblasts.

[0007] Preferably, the preparation materials of the biomimetic interpenetrating network hydrogel include sodium alginate, collagen and calcium sulfate, the final concentration of the sodium alginate is 5-20 mg / mL, the final concentration of the collagen is 1-3 mg / mL, and the final concentration of the calcium sulfate is 5-20 mM.

[0008] Preferably, the sphere culture time is 3 days.

[0009] Preferably, the myofibroblasts are reprogrammed into the cardiomyocyte-like cells, the expression of fibrosis-related genes in the cells is reduced, and the expression of cardiomyocyte-characteristic genes is increased.

[0010] More preferably, the fibrosis-related genes include at least one of Acta2, Ctgf, Col1a1 or Col3a1.

[0011] More preferably, the cardiomyocyte characteristic genes include cardiomyocyte myofiber component genes, ion channel genes or cell connection-related genes.

[0012] More preferably, the cardiomyocyte myofiber component genes include at least one of Tnni3, Myh6, Actc1 or My13, the ion channel genes include at least one of Ryr2, Kcnj2 or Kcnj12, and the cell connection-related gene is Gja1.

[0013] In a second aspect, the present application provides a preparation for improving myocardial cell fibrosis, which comprises the supernatant culture fluid of the cell spheres obtained after the sphere formation culture is completed in any of the above methods.

[0014] In a third aspect, the present application provides the use of any of the above methods in myocardial cell repair.

[0015] This application has at least the following beneficial effects:

[0016] (1) The method for achieving cell reprogramming based on a biomimetic interpenetrating network hydrogel proposed in this application does not require cell nuclear transplantation, viral transfection, or gene editing operations, and does not require the addition of additional chemical induction reagents, and is highly safe. This application preferably manufactures a biomimetic interpenetrating network hydrogel using collagen, sodium alginate, and calcium sulfate. Cell reprogramming can be achieved simply by culturing cells into spheres, with significant effects, low cost, high practicality, and huge potential for application in regenerative medicine.

[0017] (2) The method for cell reprogramming based on biomimetic interpenetrating network hydrogels proposed in this application can achieve cell reprogramming faster and more efficiently than existing methods, with the entire process taking as little as three days. By using this method, cardiomyocyte-like cells can be obtained more quickly and efficiently, reducing the number of operations and improving preparation safety, creating the prerequisite for the subsequent widespread application of reprogramming cardiac fibroblasts into cardiomyocyte-like cells.

[0018] (3) The biomimetic interpenetrating network hydrogel used in this application can mimic the viscoelastic characteristics of natural tissues. Cells can spontaneously adhere to the wall, migrate, shrink and aggregate when interacting with the biomimetic interpenetrating network hydrogel. Cell adhesion is the key to reprogramming.

[0019] (4) The method of the present application is not limited to culture vessels, which can reduce the waste of culture medium resources and is not limited by the problem of a small number of cells obtained due to the small bottom area of the culture bottle.

[0020] (5) The preparation of the present application is based on the supernatant of cell spheroids and can reverse myocardial fibrosis.

[0021] (6) The method of this application is safe and efficient, and is suitable for research and application in myocardial cell repair and improvement of myocardial fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Schematic diagram of the preparation of biomimetic interpenetrating network hydrogel and cell spheroid culture.

[0023] Figure 2 This is a characterization diagram of the mechanical properties of biomimetic interpenetrating network hydrogel.

[0024] Figure 3 Schematic diagram of the isolation and identification of primary cardiac fibroblasts.

[0025] Figure 4 Schematic diagram of the activation of primary cardiac fibroblasts into myofibroblasts cultured on stiff substrates.

[0026] Figure 5 Schematic diagram of spontaneous sphere culture of primary myofibroblasts.

[0027] Figure 6 Schematic diagram of immunofluorescence results showing the reversal and characterization of myocardial fibrosis at the cellular level.

[0028] Figure 7 Schematic diagram of sequencing analysis of mechanical reprogramming in myofibroblasts.

[0029] Figure 8 Schematic diagram of qPCR results for reversal and characterization of myocardial fibrosis at the cellular level.

[0030] Figure 9Schematic diagram of qPCR results of reprogramming cardiac fibroblasts into cardiomyocyte-like cells at the cellular level.

[0031] Figure 10 Schematic diagram of the enhanced expression of cTnI, a cardiomyocyte characteristic protein, after myofibroblast reprogramming.

[0032] Figure 11 Schematic diagram of the enhanced expression of cTnT, a cardiomyocyte characteristic protein, after myofibroblast reprogramming.

[0033] Figure 12 This figure shows the effects of drugs that inhibit cell contraction on cell morphology and gene expression.

[0034] Figure 13 Schematic diagram of senescence staining of cardiac fibroblasts from aged rats compared to those from neonatal rats.

[0035] Figure 14 This is the qPCR graph of myocardial fibrosis gene expression after culture of interpenetrating networks of cardiac fibroblasts from aged mice. DETAILED DESCRIPTION

[0036] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0037] The hydrogel in this application has both the nonlinear elastic properties of collagen and the viscoelastic properties of calcium alginate, which are similar to the mechanical characteristics of tissues, and constructs a cell culture microenvironment that mimics physiological mechanics in vitro.

[0038] The spheroid culture in this application refers to the fact that after cells are seeded on the surface of the biomimetic hydrogel in this application, the cells will spontaneously aggregate, shrink and grow until multiple cells aggregate and shrink together to form a cell spheroid form, and based on mechanical contraction, the cell volume is compressed, the cell fate is changed, and finally cell reprogramming is induced.

[0039] This application provides a method for reprogramming cardiomyocytes, comprising culturing myofibroblasts into spheres on a biomimetic interpenetrating network hydrogel. The cells spontaneously spheroidize, contract, and squeeze, causing the myofibroblasts to aggregate and reprogram the myofibroblasts into cardiomyocyte-like cells. The sphere culture period can be 3-7 days, preferably 3 days.

[0040] The biomimetic interpenetrating network hydrogel is prepared from materials including sodium alginate, collagen, and calcium sulfate. The final concentrations of sodium alginate, collagen, and calcium sulfate are 5-20 mg / mL, 1-3 mg / mL, and 5-20 mM, respectively. Cells cannot adhere to the cell wall using only sodium alginate and calcium sulfate. Cells cannot form spheres when cultured with collagen alone. If the calcium sulfate concentration exceeds the specified range, the mechanical properties of the biomimetic hydrogel will not meet the requirements of this application.

[0041] Myofibroblasts are reprogrammed into cardiomyocyte-like cells, with reduced expression of fibrosis-related genes and increased expression of cardiomyocyte-characteristic genes. Fibrosis-related genes include at least one of Acta2, Ctgf, Col1a1, or Col3a1. Cardiomyocyte-characteristic genes include cardiomyocyte myofiber component genes, ion channel genes, cell junction-related genes, and other cardiomyocyte-characteristic genes. Cardiomyocyte myofiber component genes include at least one of Tnni3, Myh6, Actc1, or My13; ion channel genes include at least one of Ryr2, Kcnj2, or Kcnj12; and the cell junction-related gene is Gja1. Other cardiomyocyte-characteristic genes include Nppa, Nppb, and others.

[0042] The reprogramming method of the present application can also be applied to reprogram liver cancer cells.

[0043] This application provides a formulation for ameliorating cardiomyocyte fibrosis. After culturing cells in an interpenetrating network hydrogel for three days, the supernatant culture fluid from the cell spheres is collected and purified by centrifugation to obtain the formulation. Experimental results have shown that incubating activated myofibroblasts with this formulation for three days can reverse cardiomyocyte fibrosis.

[0044] Example 1: Reprogramming of SD rat neonatal myofibroblasts into cardiomyocyte-like cells

[0045] A method for reprogramming cardiomyocytes, comprising the following steps:

[0046] 1. Preparation of interpenetrating network hydrogel.

[0047] (1) Add sodium alginate (Aladdin, S100128) to deionized water and stir until completely dissolved;

[0048] (2) The sodium alginate solution was dialyzed in a dialysis bag with a molecular weight cut-off of 3500d for 3 days;

[0049] (3) Filter-sterilize the sodium alginate using a 0.22 μm filter membrane and freeze-dry it;

[0050] (4) Prepare a 3.5% (w / v) solution using serum-free DMEM / F12 medium (Gibco) for later use;

[0051] (5) Type I rat tail collagen (BD354236) was prepared by mixing 10× PBS, 1 M NaOH, and ddH2O to a solution with a concentration of 3 mg / mL and a pH of 7.4;

[0052] (6) mixing the collagen solution obtained in step (5) and the sodium alginate solution in a volume ratio of 2:1;

[0053] (7) Calcium sulfate (Aladdin, A105242) was added to deionized water to prepare a suspension with a concentration of 1.22 M and sterilized by high-pressure steam;

[0054] (8) Dilute the suspension to a final concentration of 17.4 mM calcium ion solution, and mix it thoroughly with the above-mentioned collagen and alginate mixture using a syringe. Finally, place it in a 37°C cell culture incubator for 30 minutes to allow it to crosslink, and finally obtain an interpenetrating network hydrogel (calcium ion concentration 5-20 mM) with similar mechanical characteristics to in vivo tissues.

[0055] 2. Characterization of the structure and mechanical properties of biomimetic interpenetrating network hydrogels.

[0056] (1) After the interpenetrating network hydrogel was prepared, its surface structure was characterized by scanning electron microscopy (SEM). First, the interpenetrating network hydrogel was fixed with 4% paraformaldehyde for 30 minutes and washed with PBS three times, each time for 5 minutes. Then, the interpenetrating network hydrogel was dehydrated from PBS to water and then continuously transitioned to pure ethanol, that is, it was incubated in PBS, water, 30%, 50%, 70%, 90% and 100% ethanol solutions for 30 minutes respectively, and then freeze-dried. A platinum coating was sprayed on its surface, and the surface morphology and structure were observed by SEM.

[0057] (2) The mechanical characteristics of the interpenetrating network hydrogels were characterized using a rheometer MCR102. Interpenetrating network hydrogels with different calcium sulfate concentrations were made into 8 mm diameter disks and tested using an 8 mm diameter rotor after polymerization.

[0058] (3) Amplitude sweep and frequency sweep were used to test the storage / loss modulus (1 Hz, 0.1%-100% strain) and complex viscosity (0.5% strain, with a frequency of 0.1-0.5 Hz) of the interpenetrating network hydrogel. Figure 2 Show.

[0059] 3. Use bionic interpenetrating hydrogel to achieve spheroid culture and detection of SD rat neonatal myofibroblasts.

[0060] (1) Primary cells were extracted from newborn mice at day 3 of birth. Myocardial fibroblasts were first collected and purified using the differential adhesion method. The culture medium was changed every other day and the cells were frozen after the P2 generation for future use. Figure 3 shown.

[0061] (2) Cardiac fibroblasts were cultured on TCP substrate for three generations to activate and transdifferentiate into myofibroblasts, and confirmed by α-SMA immunofluorescence staining to construct a myocardial fibrosis cell model, such as Figure 4 shown.

[0062] (3) Activated myofibroblasts 4×10 5 The cells were seeded on the biomimetic interpenetrating network hydrogel in a 6-well plate and allowed to shrink spontaneously into balls. After 3 days of growth, Figure 5 As shown, Figure 5 a shows the cell growth status of myofibroblasts after being seeded on 6-well plate substrate, collagen substrate, and IPN hydrogel substrate. Figure 5 b shows the process of cell shrinkage and spheroid growth after being seeded on IPN hydrogel. Cell RNA was extracted on ice using a total RNA extraction reagent (Trizol), and the expression levels of marker genes for myofibroblast activation (Acta2, Col1a1, Col3a1, etc.) were compared with those of the control group (TCP basal culture) by qRT-PCR. Figure 7 The expression of related genes was significantly reduced after spheroid culture. At the same time, the expression of α-SMA protein was compared with the control group using WB and IF, thus verifying that the activated myofibroblasts reversed their fibrotic characteristics through three-dimensional culture of interpenetrating network hydrogels. The test results are as follows Figure 6 As shown, the results showed that the expression of myocardial fibrosis marker protein α-SMA was significantly reduced.

[0063] (4) The RNA extracted from the collected spheroid cells was used to quantitatively analyze the expression trends of myocardial cell development and differentiation genes and myocardial cell characteristic genes: myocardial cell myofiber component genes, ion channel genes, cell connection genes (Gja1), and Nppa, Nppb and other genes. The test results are as follows Figure 7-Figure 9 As shown, the results showed that the expression of genes promoting cardiomyocyte development and differentiation as well as cardiomyocyte characteristic genes was significantly enhanced, proving that myofibroblasts tend to reprogram into cardiomyocytes after IPN sphere culture.

[0064] (5) Using immunofluorescence, cardiac troponin I (cTnI) and cardiac troponin T (cTnT) were observed by confocal imaging and compared with myofibroblasts cultured with TCP. Figure 10 and Figure 11 As shown, the results showed that myofibroblasts expressed enhanced cardiomyocyte characteristic proteins after being cultured into IPN spheres.

[0065] 4. Analysis and verification of the mechanism of mechanical reprogramming of myofibroblasts by biomimetic interpenetrating network hydrogels.

[0066] (1) Different concentrations of Blebbistatin and Y-27632 were used to inhibit cell contraction. After 24 hours, live cells were stained with Calcein-AM (green fluorescent dye for live cells) to observe cell activity and morphology, confirm the role of cell contraction in the spheroidization process, and compare the gene expression of the above-mentioned group with the normal spheroidization group using qRT-PCR to verify the role of cell contraction in cell reprogramming. Figure 12 As shown, after the contraction force of cells was inhibited by IPN hydrogel, the cells were unable to form spheres, and the expression of related genes such as Actc1, Nkx2.5 and Tnni3 was reduced.

[0067] (2) The reprogramming mechanism of myofibroblasts based on mechanical perception was analyzed using mRNA sequencing, ATAC sequencing, and nuclear staining technologies. The changes and effects of related signaling pathways, transcription factors, and chromatin accessibility were explored and verified. It was confirmed that mechanical stimulation leads to changes in the nucleus and chromatin accessibility, which ultimately leads to changes in the expression of transcription factors that induce myocardial fibrosis (decreased Ctgf) and induce myocardial cell differentiation (increased Nkx2.5, Gata4, etc.), and jointly regulate the fate of myocardial fibroblasts cultured in spheres.

[0068] Example 2: Reprogramming of primary cardiac fibroblasts from aged SD rats into cardiomyocyte-like cells

[0069] A method for reprogramming cardiomyocytes, comprising the following steps:

[0070] 1. Preparation of interpenetrating network hydrogel.

[0071] (1) Dissolve sodium alginate in ultrapure water and stir overnight;

[0072] (2) Sodium alginate was dialyzed using a dialysis bag with a molecular weight cut-off of 3500d for 3 days;

[0073] (3) Sterile filtration of sodium alginate using a 0.22 μm filter membrane;

[0074] (4) Sodium alginate was freeze-dried for 3 days and re-dissolved in serum-free DMEM at a concentration of 35 mg / ml;

[0075] (5) Aliquot sodium alginate into 1.5 mL centrifuge tubes;

[0076] (6) Prepare a 3 mg / ml neutral collagen solution using 10× PBS, ultrapure water, and 1 M sodium hydroxide;

[0077] (7) Prepare 1.22 M calcium sulfate solution and sterilize by high pressure steam;

[0078] (8) Sodium alginate and collagen solution were mixed in a 1.5 mL centrifuge tube and added to a 1 mL Luer lock syringe. Figure 1 As shown in a;

[0079] (9) Dilute 1.22 M calcium sulfate with serum-free DMEM and add it to another 1 mL Luer lock syringe. Figure 1 As shown in b;

[0080] (10) Connect two syringes with a Luer lock coupler, push the syringes back and forth to mix the two solutions, and immediately deposit them into the wells of a 6-well plate. Figure 1 As shown, the 6-well plate was placed in a cell culture incubator for half an hour to solidify the collagen;

[0081] (11) The final concentrations of collagen, sodium alginate, and calcium sulfate in the hydrogel were 1.5 mg / mL, 10 mg / mL, and 15 mM, respectively;

[0082] (12) Rheological tests were performed on the hydrogel to demonstrate its viscoelastic properties.

[0083] 2. Extraction of cardiac fibroblasts from aged SD rats.

[0084] (1) 20-month-old SD rats were selected and immersed in medical disinfectant alcohol for 10 min;

[0085] (2) SD rats were killed by cervical dislocation, and the heart was quickly removed by thoracotomy, leaving sufficient length of the aorta. After the heart was isolated, it was quickly placed in 4°C EDTA buffer, at which time the heart would gradually stop beating;

[0086] (3) The heart was transferred to a perfusion buffer and rinsed appropriately, then transferred to a second perfusion buffer. The aorta was tied to the needle and the heart was perfused with perfusion buffer preheated at 37°C in a 20 ml syringe for about 2 minutes to remove air and blood. The heart was then perfused with collagenase preheated at 37°C for enzymatic hydrolysis. The hardness of the heart was checked during the process.

[0087] (4) After 30 minutes, the heart becomes soft. Cut the left and right ventricular tissues and transfer them to a clean bench. Shred the heart and add Stop Buffer to stop digestion. Use a Pasteur pipette to pipette the shredded myocardial tissue into a single-cell suspension. Filter the suspension through a 100 μm filter and aliquot into 15 ml centrifuge tubes for gravity sedimentation for 10 minutes.

[0088] (5) After gravity sedimentation, the upper layer of the tube contains non-cardiac myocytes (mainly fibroblasts) and dead cardiomyocytes, while the precipitate in the tube contains active cardiomyocytes. Centrifuge at 1000 rpm for 3 minutes, aspirate the supernatant, resuspend the precipitate in cardiac fibroblast culture medium, and seed it into a collagen culture dish.

[0089] (6) The medium was changed every 2 days until the cells were fully grown and the cells were marked as P0. After passage, β-Gal staining was used to prove that they were more senescent than the cells of the suckling mouse. Figure 13 As shown, it was then used for subsequent hydrogel culture.

[0090] 3. Use bionic interpenetrating network hydrogel to realize the contraction and spheroidization culture and detection of elderly rat cardiac fibroblasts.

[0091] Aged rat cardiac fibroblasts were digested and prepared to a final concentration of 1×10 6 The cell suspension was inoculated into the 48-well plate where the hydrogel was located, with approximately 2×10 cells / mL per well. 4 cells.

[0092] After 3 days of contact and culture with the hydrogel, a collagenase solution was prepared to separate the cells from the hydrogel. The Col1a1 gene and Acta2 gene quantitative analysis was performed on the reprogrammed aged rat cardiac fibroblasts according to the experimental requirements. The test process was the same as in Example 1. The test results are shown in FIG. Figure 14 As shown, the test results show that the method of the present application can reverse myocardial fibrosis in aged rats in vitro.

[0093] Although the present application has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications or improvements may be made to the present application. Therefore, such modifications or improvements, without departing from the spirit of the present application, are within the scope of protection claimed in the present application.

Claims

1. A method for reprogramming cardiomyocytes, characterized in that: The method comprises culturing myofibroblasts into spheres on a biomimetic interpenetrating network hydrogel, so that the myofibroblasts aggregate and reprogram the myofibroblasts into cardiomyocyte-like cells.

2. The method for reprogramming cardiomyocytes according to claim 1, wherein The myofibroblasts used before culture are in an activated state.

3. The method for reprogramming cardiomyocytes according to claim 1, wherein The preparation materials of the biomimetic interpenetrating network hydrogel include sodium alginate, collagen and calcium sulfate. The final concentration of the sodium alginate is 5-20 mg / mL, the final concentration of the collagen is 1-3 mg / mL, and the final concentration of the calcium sulfate is 5-20 mM.

4. The method for reprogramming cardiomyocytes according to claim 1, wherein The sphere culture time is 3 days.

5. The method for reprogramming cardiomyocytes according to claim 1, wherein The myofibroblasts are reprogrammed into the cardiomyocyte-like cells, the expression of fibrosis-related genes in the cells is reduced, and the expression of cardiomyocyte characteristic genes is increased.

6. The method for reprogramming cardiomyocytes according to claim 5, characterized in that: The fibrosis-related genes include at least one of Acta2, Ctgf, Col1a1 or Col3a1.

7. The method for reprogramming cardiomyocytes according to claim 5, characterized in that: The cardiomyocyte characteristic genes include cardiomyocyte myofiber component genes, ion channel genes or cell connection-related genes.

8. The method for reprogramming cardiomyocytes according to claim 7, characterized in that: The myocardial cell myofiber component genes include at least one of Tnni3, Myh6, Actc1 or My13, the ion channel genes include at least one of Ryr2, Kcnj2 or Kcnj12, and the cell connection-related gene is Gja1.

9. A preparation for improving myocardial cell fibrosis, characterized in that: The method comprises the supernatant culture fluid of the cell spheres obtained after the sphere formation culture is completed in the method according to any one of claims 1 to 8.

10. Use of the method according to any one of claims 1 to 8 in repairing myocardial cells.

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