A method of inducing differentiation of induced pluripotent stem cells into sinoatrial node pacemaker cells

Abstract

A novel method for inducing differentiation of induced pluripotent stem cells into sinoatrial node pacemaker cells, by adding small molecule compounds U0126 and RA in the culture medium during in vitro differentiation process to control the differentiation direction of iPS cells, taking Shox2 as a marker for sinoatrial node head pacemaker cells, and taking Shox2 and Nkx2.5 as markers for sinoatrial node junction and intermodal bundle pacemaker cells to monitor the differentiation process of cells, can effectively induce two pacemaker cell subtypes with uniform phenotype and consistent electrophysiological characteristics. The study of directional induction of mouse iPS cells into two pacemaker cell subtypes provides a research basis for exploring the directional induction of human iPS cells into two pacemaker cell subtypes, screening small molecule compounds that can promote the directional differentiation of iPS cells into specific types of pacemaker cells, and providing uniform, stable and reliable stem cell sources for the preparation of biological pacemakers.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of stem cell technology and molecular genetics, and particularly relates to a method for differentiating induced pluripotent stem cells into sinoatrial node pacemaker cells. BACKGROUND

[0002] With the development of stem cell technology and molecular genetics, especially somatic cell reprogramming technology, the source of seed cells for constructing biological pacemakers for treatment and tissue repair has been fundamentally solved. At present, the key to constructing a biological pacemaker is to keep its pacemaker activity consistent with the normal physiological pacemaker of the human body. Using somatic cell reprogramming technology, researchers can induce iPS cells to differentiate into specific cardiac conduction cell types after forming embryoid bodies, or directly overexpress certain lineage-specific transcription factors in terminally differentiated somatic cells, thereby directly reprogramming the cells into the desired cell type.

[0003] In most past studies, the cells formed by differentiation of iPS cells or direct reprogramming are mostly mixed cell populations with heterogeneous phenotypes and physiological characteristics, and most of the cells have the characteristics of ventricular cardiomyocytes. Therefore, establishing an effective method for inducing iPS cells to differentiate into cardiac conduction system pacemaker cells can help further study the development of the cardiac conduction system and the occurrence of related diseases, and can also provide a reliable cell source for developing biological pacemakers. The results of Ye et al. showed that the sinoatrial node of humans and mice is composed of a head region expressing only Shox2 and Hcn4, and another common sinoatrial junction region expressing Shox2, Nkx2.5 and Hcn4. The study of Li et al. further confirmed that there are two types of pacemaker cells with different electrophysiological properties in the sinoatrial node, i.e., Shox2 + pacemaker cells and Shox2 + / Nkx2.5 + pacemaker cells.

[0004] To distinguish these two cell subtypes, the inventors of the present application isolated fibroblasts without fluorescence from E12.5 Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26R mT / mG mouse embryos, and introduced the Shox2 Oct4 , Klf4 , c- Myc and Sox2The four embryonic stem cell specific genes are infected into fibroblasts by lentivirus, and then the iPS cells are added into 96-well low-adhesion round-bottom plates to form embryoid bodies for spontaneous differentiation. The results show that the iPS cells also have the potential to differentiate into Shox2-expressing cells and Shox2 and Nkx2.5 co-expressing cells, and the two types of pacemaker cells induced by the method have similar electrophysiological characteristics to the pacemaker cells in vivo. However, the low efficiency of the embryoid body differentiation into pacemaker cells is a problem. The iPS cells are differentiated into Shox2 + Pacemaker cell yield is only 8.3%, Shox2 + / Nkx2.5 + Pacemaker cell yield is only 4%. Therefore, how to improve the efficiency of iPS cell induction into pacemaker cells has become one of the focuses of the previous research. SUMMARY

[0005] In order to overcome the deficiencies of the prior art, the present application provides a new method for differentiating induced pluripotent stem cells into sinoatrial node pacemaker cells, which controls the differentiation direction of iPS cells by adding different small molecule compounds in the culture medium during in vitro differentiation, and uses Shox2 as a marker for sinoatrial node head pacemaker cells, and Shox2 and Nkx2.5 as markers for sinoatrial node junction and intermodal bundle pacemaker cells to monitor the differentiation process of the cells, which can effectively induce two subtypes of pacemaker cells with uniform phenotype and consistent electrophysiological characteristics.

[0006] The technical solution adopted by the present application is a new method for differentiating induced pluripotent stem cells into sinoatrial node pacemaker cells, which comprises the following steps:

[0007] (1) Isolation and culture of mouse embryonic fibroblasts;

[0008] (2) Reprogramming of mouse MEF into mouse iPS cells by STEMCCA lentivirus infection reagent;

[0009] (3) iPS cell clone colony selection;

[0010] (4) iPS cell resuscitation;

[0011] (5) iPS cell differentiation to obtain embryoid bodies (EB);

[0012] (6) Inducing differentiation of embryoid bodies (EB) by small molecule compounds U0126 and RA to obtain Shox2 + and Shox2 + / Nkx2.5 + Pacemaker cell subtypes;

[0013] (7) Patch clamp technique is used to detect the electrophysiological characteristics of the differentiated pacemaker-like cells.

[0014] The working concentration of compound U0126 in step (6) is 10 μM, and the working concentration of compound RA is 1 μM.

[0015] The time node of adding the small molecule compounds U0126 and RA in step (6) is the second day of EB differentiation.

[0016] The isolation and culture of the mouse embryonic fibroblasts in step (1) comprises the following steps:

[0017] (a) Take E12.5 mouse embryos, move the fetal mice into a sterile culture dish pre-cooled with DPBS, and remove the head, limbs, internal organs and parts with fluorescent protein markers of the fetal mice with ophthalmic scissors and ophthalmic forceps;

[0018] (b) Move the remaining mouse embryo tissues to a new DPBS culture dish, and wash the tissues with DPBS three times until there is no visible blood color;

[0019] (c) Put the mouse embryo tissues into a centrifuge tube, and add 0.5 mL of 0.25% trypsin-EDTA digestive solution to each centrifuge tube;

[0020] (d) After repeatedly blowing the tissues with a pipette gun for about 20 times, place the centrifuge tube in a 37°C incubator for 3 min, add MEF culture medium to terminate digestion, centrifuge for 5 min, aspirate the supernatant, add 1 mL of MEF culture solution to resuspend the cell pellet in each centrifuge tube, and inoculate it into a 12-well plate, and place it in a 37°C, 5% CO2 incubator for culture, and change the medium every day;

[0021] (e) Obtain Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26R mT / mG The mouse is a mouse that will label the cell membrane expressing Nkx2.5 with GFP green fluorescence, and the cells expressing Shox2 can be labeled with LacZ live cell dye, after obtaining the fetal mouse heart, cut the mouse heart into two parts along the coronary section with ophthalmic scissors, and under a fluorescence microscope, use sharp forceps and a 32G needle to completely separate and collect the Shox2 + head cells of the sinoatrial node, Shox2 + / Nkx2.5 + sinoatrial node junction cells; digest them into single cells using collagenase type II, and then culture them; after the cells adhere, detect the electrophysiological characteristics of the cells using whole-cell patch clamp technique;

[0022] (f) After the wild type E12.5, E14.5, E16.5 fetal mouse, 2-day-old and 4-week-old mouse heart were sliced using a vibrating microtome, the patch clamp technique was used to detect the pacing characteristics of the cells.

[0023] The step (2) of reprogramming mouse MEF into mouse iPS cells by STEMCCA lentivirus infection reagent includes the following steps:

[0024] (I) Day 0, each well was coated with 2 mL of 0.1% gelatin solution in a sterile 6-well plate. Incubate in a 37°C incubator for at least 30 minutes, discard the gelatin solution, and plate the 6-well plate with a density of 1 x 10 5 Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26R mT / mG The mouse MEF was added with 3 mL of fresh culture medium, and the cells were placed in a 37°C, 5% CO2 incubator overnight; Day 1, discard the old culture medium, add 3 mL of fresh MEF culture medium. Add 1.5 μL of Polybrene transfection reagent to each well. Place the plate in a 37°C, 5% CO2 incubator until the lentivirus is ready to be added;

[0025] (II) Thaw 1 tube of Mouse STEMCCA Constitutive Polycistronic (OKSM) lentivirus at room temperature, and immediately place it on ice after thawing. Add the required volume of lentivirus according to the following formula:

[0026]

[0027] Note: The MOI value and Virus Titer of this lentivirus reagent are calculated by Millipore Company;

[0028] (III) After adding the required volume of virus to the well containing the attached MEF, gently shake the six-well plate in a cross shape to evenly cover the MEF with the lentivirus, and place the six-well plate in a 37°C, 5% CO2 incubator overnight;

[0029] (IV) Day 2, discard the old culture medium, and replace it with complete iPS cell culture medium containing 15% FBS and LIF; Day 3 - Day 15, replace the fresh iPS cell culture medium every day, and the mouse iPS cell clones will generally appear in 10-13 days.

[0030] The specific steps of step (3) of iPS cell colony selection are as follows:

[0031] ​(1) After the iPS cell clone colony grows to the appropriate size, the 24-well plate is covered with 0.1% gelatin one day before the iPS cell clone colony is selected.

[0032] (2) The iPS cells are examined under an inverted microscope, and the appropriate cell colony is selected. A 10 μL gun tip is used to scratch around the target colony, and the cell clone colony is scraped off with a pipette tip. The cell colony is then sucked into the gun tip.

[0033] (3) The cell clone colony is transferred to a 96-well plate, 100 μL of 0.25% Trypsin-EDTA is added to each well, and incubation is performed at 37°C for 2 minutes. The digestion is terminated with an equal volume of iPS cell culture medium, and the cells are gently blown into a single-cell suspension with a 100 μL pipette.

[0034] (4) The gelatin in the 24-well plate is discarded, and the resuspended iPS cells are transferred to a 24-well plate and 1 mL of fresh iPS cell culture medium is added. The 24-well plate is incubated in a 37°C, 5% CO2 incubator, and the medium is changed daily. Generally, the iPS cells can be passaged after growing in the 24-well plate for 7-10 days.

[0035] The specific steps of step (4) iPS cell resuscitation are as follows:

[0036] (1) At least half an hour in advance, 0.1% Gelatin is added to the six-well plate, 1 mL per well, to cover the bottom. Then the 6-well plate is placed in a 37°C incubator for more than 30 min.

[0037] (2) Confirm that the water bath temperature is maintained at 37°C in advance, and take out the culture medium to warm it up.

[0038] (3) Take the cells out of the liquid nitrogen tank and place them in a -80°C pre-cooled cryopreservation box or dry ice. Then as soon as possible, place the cryopreservation tube in a 37°C water bath for rapid resuscitation. Do not shake the tube, place it on the centrifuge tube rack in the water bath, and wait for the cell liquid to melt. At the same time, the six-well plate can be taken out, the Gelatin is discarded, and 2 mL of fresh culture medium is added to each well.

[0039] (4) After the cell liquid has completely melted, centrifuge the cryopreservation tube in the centrifuge at 300 x g for 5 min. g

[0040] (5) Discard the supernatant, leave 100 μL of supernatant, and add 1 mL of fresh culture medium to mix the cells. Blow the cells for about 5 times.

[0041] (6) Add the mixed cell liquid to the culture medium, and the resuscitation is complete.

[0042] ​The step (5) iPS cell differentiation to obtain the embryoid bodies, and the specific steps are as follows:

[0043] (I) When the growth density of the ES or iPS cells reaches about 80%, the cells are digested and blown into a single cell suspension by using 0.25% trypsin-EDTA;

[0044] (II) The cells are inoculated into a culture dish coated with 0.1% gelatin at a certain density, and then placed in a 37°C 5% CO2 incubator for 30 min. The unadherent cells in the supernatant are removed, and the cells are centrifuged at 300 x g for 5 min; g

[0045] (III) The cells are resuspended in the embryoid body differentiation medium, and then inoculated into an Ultra Low Attachment 96-well plate for suspension drop culture. 1000 cells are added to each well, and 300 μL of the culture solution is added;

[0046] (IV) After 24 h of culture, granular globules can be observed under a microscope, which are the embryoid bodies (EB). The EB globules can emit spontaneous beats after about 7 d of continuous culture.

[0047] The step (7) patch clamp technique for detecting the electrophysiological characteristics of the differentiated pacemaker-like cells, and the specific steps are as follows:

[0048] (1) Glass electrode drawing: import a glass blank with a core (its inner diameter is 0.86 mm, and its outer diameter is 1 mm), and the electrode is drawn as needed.

[0049] (2) The tip of the electrode is about 1-2 μm, the prepared intracellular solution is filled in the microelectrode, and the detection impedance is about 5-10 MΩ.

[0050] (3) The cover glass with cells is placed in the perfusion tank of the experimental operation table, and kept at a constant temperature of 37°C. The electrode external solution is slowly dropped, and 95% oxygen and 5% carbon dioxide are continuously introduced into the extracellular solution.

[0051] (4) The front end of the prepared microelectrode is filled with a proper amount of electrode internal solution without bubbles, which is sleeved outside the silver inner core of the electrode. Under a microscope, the tip of the glass electrode is found, the microelectrode tip is slightly touched on the surface of the cells, and a small amount of negative pressure suction is given to promote the contact between the electrode and the cell membrane. The current square wave and resistance change are observed on the display interface of the amplifier. If the resistance value exceeds and stabilizes at more than GΩ, it is proved that the electrode and the cell form a stable high-order block.

[0052] ​(5) Microelectrode inner micro negative pressure to absorb broken cell membrane, so that the electrode and intracellular liquid liquid communication, and then clamp voltage stable at -70 mV, to current clamp mode, by clamping membrane current to record the change of transmembrane voltage, then use pCLAMP software acquisition, storage and analysis of signals.

[0053] The beneficial effects of the present application are: the present application provides a novel method for inducing differentiation of induced pluripotent stem cells into sinoatrial node pacemaker cells, by adding small molecule compounds U0126 and RA in the culture medium during in vitro differentiation process to control the differentiation direction of iPS cells, taking Shox2 as a marker of sinoatrial node head pacemaker cells, and taking Shox2 and Nkx2.5 as markers of sinoatrial node junction and intermodal bundle pacemaker cells to monitor the differentiation process of cells, which can effectively induce two pacemaker cell subtypes with uniform phenotype and consistent electrophysiological characteristics. The research on the directional induction and differentiation of mouse iPS cells into two pacemaker cell subtypes provides a research basis for exploring the directional induction and differentiation of human iPS cells into two pacemaker cell subtypes and screening small molecule compounds that can promote the directional differentiation of iPS cells into specific type pacemaker cells, and provides a uniform, stable and reliable stem cell source for preparing a biological pacemaker. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 To Nkx2.5 IRESCre / + ; Shox2 LacZ / + ;ROSA26 mG fibroblasts into iPS cells, and identify its biological characteristics; wherein A a: Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG Morphological characteristics of iPS cells. Ab-c: in Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / m G Expression of markers specific to embryonic stem cells in iPS cells Oct3 / 4 , Ssea4 and Nanog . B: detection of stem cell pluripotency markers and reprogramming transcription factors Oct3 / 4 , Klf4 , Sox2 , C-myc and pluripotent stem cell markers Nanog in Nkx2.5 IresCRE / + ;Shox2 LacZ / + ; ROSA26 mT / mG and WT mouse fibroblasts, Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG and iPS cells of WT mice, G4 embryonic stem cell line and their 15-day differentiated embryoid bodies. Scale bar: 50 μm.

[0055] Figure 2 Expression levels of stem cell pluripotency related genes and cardiac lineage markers during embryoid body differentiation.

[0056] Figure 3 To identify Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG whether iPS cells have the potential to differentiate into two subtypes of pacemaker cells; wherein A: after 7 days of differentiation, Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG iPS cell differentiated EBs are able to spontaneously beat and express green fluorescent protein that can track Nkx2.5 expression. B-E: identification of Shox2, Nkx2.5 and Hcn4 expression in differentiated EBs. Scale bar: 50 μm.

[0057] Figure 4 Comparison of electrophysiological properties of iPS cell differentiated Shox2+, Shox2+ / Nkx2.5+ cells and in vivo cells; wherein A: whole-cell patch clamp technique to detect action potentials of Shox2+, Shox2+ / Nkx2.5+ cells differentiated from iPS cells and in vivo cells. B: statistics of Shox2 + , Shox2+ / Nkx2.5+ and Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ;ROSA26 mT / mG Shox2 + / Nkx2.5 +The parameters of cellular action potentials, including Amplitude, Distolic duration, Distolic slope, Vmax (rate of rise of action potential), APD50 (50% action potential duration), APD90 (90% action potential duration), and APD50 / 90 (ratio of 50% action potential duration to 90% action potential duration), were calculated and plotted by independently repeating the experiment five times, using Mean ± SEM.

[0058] Figure 5 Screening can promote Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ;ROSA26 mT / m G iPS cells differentiate into Shox2 cells. + With Shox2 + / Nkx2.5 + Small molecule compounds for pacemaker cells; A: Schematic diagram of inducing cell differentiation into pacemaker cells by adding small molecule compounds. B: Optimal working concentrations of small molecule compounds were determined using trypan blue staining combined with automated cell technology instruments. The optimal working concentrations for each small molecule compound were: BIO, 3 μM; RA, 1 μM; A83-01, 1 μM; Suramin, 0.5 mM; U0126, 10 μM; SB203580, 0.1 μM; VPA, 500 mM. C: On day 20 of differentiation, the number of EB cells that maintained pacing after treatment with different small molecule compounds was counted. The experiment was independently repeated 3 times, and the results were calculated and plotted according to Mean ± SEM. * indicates statistically significant differences between groups, ** p <0.05,** p <0.01.

[0059] Figure 6 To enable real-time qPCR detection of the expression of cardiac lineage-related biomarkers induced by the addition of small molecule compounds.

[0060] Figure 7 Small molecule compounds screened by flow cytometry were used to induce iPS cell differentiation into Shox2 cells. + and Shox2 + / Nkx2.5 +Cell efficiency; A: Flow cytometry cell sorting results for BIO, RA, U0126, VPA, and Suramine. B: Statistical graph of flow cytometry cell sorting results for BIO, RA, U0126, VPA, and Suramine. Experiments were independently repeated at least three times. Statistical analysis and plotting were performed using mean ± SEM combined with one-way ANOVA. p <0.01.

[0061] Figure 8 To detect the expression of heart-related genes in cells sorted by flow cytometry using RT-PCR. Figure 9 Electrophysiological characteristics of RA-induced Shox2+ / Nkx2.5+ cells and U0126-induced Shox2+ cells were recorded. A: Action potentials of RA-induced Shox2+ / Nkx2.5+ cells and U0126-induced Shox2+ cells were detected using whole-cell patch-clamp technique. B: Total potassium currents of RA-induced Shox2+ / Nkx2.5+ cells and U0126-induced Shox2+ cells were recorded. The clamping voltage was -35 mV, the stimulation time was 1.5 s, and the voltage pulse was increased from -105 mV to +35 mV in 10 mV increments to activate the total potassium current. C: Shox2+ cells were statistically analyzed in the head of the sinoatrial node, the sinoatrial node junction, and the internodal tracts of E13.5 mouse embryos. + Parameters of action potentials in Shox2+ / Nkx2.5+ and RA-induced Shox2+ / Nkx2.5+ cells and U0126-induced Shox2+ cells, including Amplitude, Distolic duration, Distolic slope, Vmax (rate of rise of action potential), APD50 (50% action potential duration), APD90 (90% action potential duration), and APD50 / 90 (ratio of 50% to 90% action potential duration). D: Current-voltage curves plotted based on the relationship between current and voltage in RA-induced Shox2+ / Nkx2.5+ and U0126-induced Shox2+ cells. Statistical analysis and plotting were performed using SEM combined with one-way ANOVA, * p <0.05,** p <0.01, *** p <0.001, n = 7.

[0062] Figure 10 Gel electrophoresis images of total RNA extracted from each sample. Figure 11Sample clustering plot of the transcriptome data of the control group and the experimental group treated with U0126; wherein the horizontal and vertical coordinates of the figure are the names of each sample, and the color from dark to light represents the correlation between samples from strong to weak. Figure 12 Heat map of genes with differential expression after treatment with U0126.

[0063] Figure 13 Functional enrichment clustering analysis of genes with up-regulated expression after treatment with U0126; wherein the horizontal coordinate of the figure is the sample grouping name, and the vertical coordinate is the GO annotation Biology process functional enrichment. The color from dark to light represents the value size, and the circle size represents the number of genes with GO function annotation. q

[0064] Figure 14 Verification of the mRNA expression results of part of the differential genes. DETAILED DESCRIPTION

[0065] The present application is further illustrated in conjunction with the accompanying drawings and the following embodiments, which should be understood as merely illustrating the present application, rather than limiting the present application.

[0066] Instrument and equipment:

[0067]

[0068] Experimental animals:

[0069] The SPF C57BL / 6, ICR background mice and nude mice used in the experiments in this chapter were ordered from Shanghai Slac Animal Laboratory Co., Ltd. The 6-week-old or older mice were used in the experiments. Nkx2.5 IRESCre / + The mice were crossed to obtain ROSA26R mT / mG Nkx2.5 IRESCre / + ; ROSA26R mT / mG The genotype mouse offspring, and then mating the mouse with Shox2 LacZ / + mouse and breeding, finally obtained Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26R mT / mG mouse.

[0070] Experimental reagents:

[0071]

[0072] Reagent preparation:

[0073] ​​(1) Mouse Embryonic Fibroblast (MEF) Medium (500 mL) and ESC, iPS Cell Medium Preparation

[0074]

[0075] After the preparation of the Mouse Embryonic Fibroblast Medium, the medium is filtered with Nalgene Rapid-Flow Sterile Disposable Filter Units with PES Membrane and stored at 4°C until use. The Mouse ESC / iPS Medium is prepared by adding ESGRO mLIF Medium Supplement at a ratio of 1:500 to the Fibroblast Medium to inhibit differentiation of the cells.

[0076] (2) Embryoid Body Differentiation Medium

[0077]

[0078] (1) 0.1% Gelatin

[0079] Dissolve 0.1 g of Gelatin in 100 mL of deionized water, autoclave, filter with a 0.22 μm filter and store at 4°C until use.

[0080] (2) Preparation of reagents in the FACS blue LacZ β-galactosidase detection kit

[0081] Dilute the stock solution of Blue LacZ staining substrate reagent (50 mM) in 2 mL of reaction buffer to a working concentration of 10 mM. Store in the dark at -20°C. The working concentration of the substrate reagent after aliquoting should be protected from repeated freeze-thaw cycles. The diluted staining substrate reagent can be stored at -20°C for at least 6 months.

[0082] Prepare 50 mL of 200 mM sodium phosphate buffer (2X) by mixing 30.5 mL of 200 mM Na2HPO4with 19.5 mL of 200 mM NaH2PO4, adjusting the pH to 7.0. Then add 1 mM MgCl2, 10 mM β-mercaptoethanol and 0.1% Triton X-100 to the sodium phosphate buffer and dilute to a final volume of 100 mL with ddH2O.

[0083] In previous studies we have shown that from the constructed Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26RmT / mG In mice, pacemakers at the sinoatrial node head expressing only Shox2 and pacemakers at the sinoatrial node junction co-expressing Shox2 and Nkx2.5 can be completely separated. Cells expressing Nkx2.5 are labeled with GFP green fluorescence, while cells expressing Shox2 carry the β-galactosidase reporter gene. LacZ Using a β-galactosidase-active dye, cells expressing Shox2-LacZ exhibited blue fluorescence upon ultraviolet light excitation, making them clearly identifiable under a fluorescence microscope. Patch-clamp techniques further revealed that these cells possess unique pacemaker cell electrophysiological characteristics in vivo, demonstrating that cardiac pacemakers have at least two subtypes. [6] To obtain a large number of pacemaker cell subtypes with uniform phenotype and electrophysiological properties in vitro, and to provide stem cell support for the future development of biological pacemakers to treat cardiac conduction system dysfunction, we started with E12.5. Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26R mT / mG Cells that do not express green fluorescent protein (GFP) were isolated from mouse embryos. Since GFP expressed by Nkx2.5 is continuously expressed once activated, we needed to remove fibroblasts that already expressed GFP to better observe Nkx2.5 expression during iPS cell differentiation. After obtaining the fibroblasts, we followed the instructions in the iPS cell induction kit. [The text then abruptly shifts to a seemingly unrelated topic:] ...and then... Oct4 , Klf4 , c-Myc and Sox2 Lentiviral infections of fibroblasts with four embryonic stem cell-specific transcription factors (OKSM) induced a reprogramming process. After 10 days, fibroblast morphology underwent significant changes, exhibiting clonal colony formation resembling that of embryonic stem cells. Once the colonies continued to grow and formed clear boundaries, they were selected and cultured and expanded independently in 96-well plates. These cells grew in clonal colonies, exhibiting uniform morphology, tight arrangement, high nucleoplasm-to-cytoplasm ratio, and prominent nucleoli, closely resembling embryonic stem cells (e.g., ...). Figure 1 A a).

[0084] To verify that the harvested cells were successfully reprogrammed into iPS cells, we used immunofluorescence staining to detect the biological characteristics of the established iPS cells. Immunofluorescence staining results showed that the expression of pluripotency-related transcription factors Oct4 and Nanog, and the stem cell-specific surface marker protein Ssea4, could be detected in the established iPS cells. Figure 1A b-c). The results show that the iPS cells we harvested have similar biological characteristics to embryonic stem cells.

[0085] Since the reprogramming process is initiated by the exogenous reprogramming factor OKSM to express endogenous pluripotency-related genes to obtain iPS cells. When the endogenous factor is activated, the exogenous OKSM needs to be inactivated to avoid affecting the differentiation of iPS cells. Next, we will use RT-PCR (reverse transcription) to detect the inactivation of Oct4 、 Sox2 、 Klf4 and c- Myc . At the same time, since we choose Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26R mT / mG Mice are transgenic mice, in order to test the effect of the gene we knocked in on the differentiation process of iPS cells, we use G4 embryonic stem cell line (C57 / BL6 background wild type mouse embryonic stem cell line) and iPS cells reprogrammed from skin fibroblasts taken from E12.5 wild type mice (WT) as controls, to detect the expression of Oct4 、 Sox2 、 Klf4 、 c-Myc and endogenous transcription factor Nanog in MEF (fibroblasts) before reprogramming, iPS cells after reprogramming and EBs formed by differentiation. Figure 1 B shows that all the WT and Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26R mT / mG iPS cells we established successfully activated the expression of endogenous reprogramming transcription factor Nanog , and the expression of Oct4 、 Sox2 、 Klf4 、 c-Myc in EBs formed by differentiation of each cell was basically inactivated, which further proves that we successfully constructed Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26R mT / mG iPS cells.

[0086] As shown in Figure 1 , the morphology observation, immunofluorescence staining and RT-PCR were used to establish Nkx2.5IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG The biological characteristics of iPS cells were detected, with G4 embryonic stem cell line (wild type mouse embryonic stem cell line in C57 / BL6 background) and skin fibroblasts taken from E13.5 wild type mice (WT) reprogrammed to form iPS cells as controls.

[0087] To further verify the genetically modified iPS cells Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG The differentiation process of iPS cells is not affected by the knock-in gene. We continued to detect the expression changes of pluripotency marker genes Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG iPS cells during differentiation Oct4 , Nanog and Rex1 mesoderm marker genes Mesp1 , pacemaker cell related genes Shox2 and Hcn4, cardiac progenitor cell marker genes Nxk2.5 and working cardiomyocyte marker genes Mef2c . In this study, we used the method of adding 500-1000 iPS cells s to a low-adhesion 96-well round-bottom plate to induce iPS cells to spontaneously differentiate by aggregating and forming embryoid bodies (EBs)

[16] . The whole induction and differentiation culture process is 30 days, and the 2, 5, 8, 15, and 30 days of differentiation are selected for Real-time qPCR detection of the expression of the above genes. As Figure 2 shown, the results show that these genes change similarly in iPS cells and control groups, indicating Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG The expression change rules in iPS cells and control groups are similar, indicating Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG The differentiation process of iPS cells is not affected by the knock-in gene. In summary, we successfully established iPS cells with similar biological characteristics and differentiation ability to embryonic stem cells and WT iPS cellsNkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG iPS cells.

[0088] Figure 2 G4 ESCs, WT and Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG iPS cells were tested for expression of stem cell pluripotency related genes Oct3 / 4 , Nanog , Rex1 mesodermal lineage factors Mesp1 , transcription factors regulating the differentiation direction of cardiac pacemaker cells Shox2 , cardiac conduction system pacemaker markers Hcn4 , cardiac progenitor markers Nkx2.5 and mature cardiomyocyte markers Mef2c during differentiation. Experiments were repeated independently 3 times and calculated and plotted as Mean ± S.E.M.

[0089] To test whether the differentiated Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG iPS cells have the potential to differentiate into both pacemaker cell subtypes, we tested the expression of Shox2, Nkx2.5 and Hcn4 in the EBs after 7 days of differentiation by immunofluorescence. As shown in Figure 3 , the differentiated EBs were beating after 7 days ( Figure 3 A), and both cells expressing only Shox2 and Hcn4 and cells co-expressing Shox2, Nkx2.5 and Hcn4 were found in the differentiated EBs. This result indicates Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG iPS cells do have the potential to differentiate into both pacemaker cell subtypes.

[0090] To verify that the differentiated Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ;ROSA26 mT / mGTo determine whether iPS cell-derived Shox2+ and Shox2+ / Nkx2.5+ cells have similar electrophysiological properties to pacemaker cells in vivo, we used patch clamp technique to measure the action potential of these two types of cells. As shown in Figure 4 Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG iPS cell-derived Shox2 + , Shox2 + / Nkx2.5 + cells have similar electrophysiological properties to Shox2 + , Shox2 + / Nkx2.5 + cells in vivo. This result indicates that iPS cell-derived differentiated cells have consistent electrophysiological properties with cells in vivo.

[0091] In the above experiments, we successfully established a method for inducing iPS cells into pacemaker cells, solving the problem of cell source. We also established an in vitro model for monitoring the differentiation of iPS cells into two subtypes of pacemaker cells. However, there is still a problem of low efficiency in using embryoid bodies to differentiate into pacemaker cells. The yield of iPS cell differentiation into Shox2+ pacemaker cells is only 8.3%, and the yield of Shox2+ / Nkx2.5+ cells is only 4%. Therefore, how to improve the efficiency of iPS cell induction into pacemaker cells has become the focus of our research.

[0092] According to the articles reported, we selected small molecule compounds BIO, A83-01, Retinoic Acid (RA), Suramin, U0216, SB203580, and VPA that can induce cells to differentiate into cardiomyocytes and pacemaker cells. Figures 3-5 A shows the time node of adding these small molecule compounds. Then we need to screen the best concentration of these small molecule compounds. Using trypan blue staining combined with automatic cell technology instrument to detect the survival rate of cells treated with different concentrations of small molecule compounds (as shown in Figure 5 B). Finally, we confirmed the best working concentration of each chemical small molecule compound as follows: BIO, 3 μM; RA, 1 μM; A83-01, 1 μM; Suramin, 0.5 mM; U0126, 10 μM; SB203580, 0.1 μM, VPA, 500 mM. Then we counted the number of EBs that kept beating after EB differentiation for 20 days after treatment with different small molecule compounds ( Figure 5 ​C). The results showed that, compared with the control group (culture medium with 1% DMSO added), only VPA could significantly promote the differentiation of iPS cells into beating cells.

[0093] To further screen small molecule compounds, we used quantitative real-time qPCR to detect EB differentiation at day 20. Shox2 , Hcn4 , Nkx2.5 , Tbx3 , Cx40 , α-SA , Islet1 The expression. For example... Figure 6 As shown, the results indicate that BIO, RA, and U0126 can all promote the differentiation of iPS cells into cardiac lineage cells and can promote the expression of Hcn4, a marker for inducing pacemaker cells in the cardiac conduction system. Although VPA did not promote the expression of Hcn4 and Shox2, it significantly increased the number of beating EBs. Therefore, we selected BIO, RA, U0126, and VPA for further experimental optimization.

[0094] Next, we used flow cytometry to analyze the efficiency of the screened small molecule compounds in inducing iPS cell differentiation into two pacemaker cell subtypes. For example... Figure 7 As shown, after screening, we found that U0126 improved the differentiation of iPS cells into Shox2 cells. + The efficiency of pacemaker cells was 28.5%, while RA improved the differentiation of iPS cells into Shox2 cells. + / Nkx2.5 + Pacemaker cells (11.8%) and Nkx2.5 + The efficiency of cardiomyocytes increased (from 10.2% to 28.1%). The results indicate that RA can promote... Nkx2.5 IRESCcre / + ; Shox2 LacZ / + ; ROSA26 mT / mG Shox2 cells differentiate more into iPS cells + / Nkx2.5 + And Nkx2.5 + U0126 can promote the differentiation of iPS cells into more Shox2 cells. + cell.

[0095] To verify the differentiation of Shox2 induced by small molecule compounds + and Shox2 + / Nkx2.5 + We used RT-PCR to detect genes specific to pacemaker cells to characterize their biological properties. Shox2 ,Hcn4 , Tbx3 , pacemaker cell genes specific to the head of the sinoatrial node Tbx18 , markers of cardiac progenitor cells Nkx2.5 and working cardiomyocytes. The results show that Shox2 + cells induced to differentiate by U0126 specifically express only markers of sinoatrial node head pacemaker cells Tbx18 , but not markers of Nkx2.5 and working cardiomyocytes Cx40 ( Figure 8 ). This result shows that Shox2 + cells induced to differentiate by U0126 have similar biological properties to Shox2+ pacemaker cells in vivo, whereas Shox2 + / Nkx2.5 + cells induced by RA have similar biological properties to Shox2 + / Nkx2.5 + pacemaker cells in vivo.

[0096] To verify the electrophysiological properties of Shox2 + / Nkx2.5 + cells induced by RA and Shox2+ cells induced by U0126. We examined their action potentials using patch clamp techniques, as shown in Figure 9 , the electrophysiological properties of Shox2+ cells induced by U0126 and Shox2 + / Nkx2.5 + cells induced by RA are similar to the electrophysiological properties of Shox2 + , Shox2 + / Nkx2.5 + cells in vivo, respectively. This result shows that cells derived from iPS cells can be effectively differentiated into pacemaker cell subtypes with uniform phenotypes and electrophysiological properties after induction by small molecules and screening by specific molecular markers. These results are expected to provide experimental evidence for efficient generation of pacemaker cell subtypes with uniform phenotypes and electrophysiological properties from any iPS cell line in the future.

[0097] Quality assessment of the transcriptome sequencing results

[0098] To further study ERK / MAPK kinase signaling pathways ERK1 / 2 inhibitors U0126

[13] ​The effect of iPS cell differentiation to pacemaker cells was analyzed by RNA-seq on EBs treated with U0126 and differentiated to 11 days, with EBs treated with 1% DMSO and differentiated to 11 days as a Control group, and two biological replicates were designed. After RNA extraction, gel electrophoresis analysis showed that each RNA sample had 18S, 28S and 5S RNA bands Figure 10 ), indicating that the extracted RNA was of good quality and no degradation occurred. Therefore, the samples were sent to Novogene Sequencing Company for library construction and sequencing analysis.

[0099] The company used Illumina HiSeq2500 sequencer for sequencing, and each sample was subjected to 8G data detection. The filtered sequencing results are shown in Table 1, and the proportion of raw data Clean Reads was more than 99%. The results of Phred (Phred Score, Qphred) value calculation for four samples showed that the quality value of each base of the four samples was more than 51. The percentage of Q30 was about 90%. These results showed that the sequencing quality of the four samples was good, and the sequencing quality met the requirements of downstream analysis.

[0100] Table 1 RNA-seq data quality of each sample

[0101]

[0102] Differential gene analysis of cells induced by U0126 and control cells

[0103] The four transcriptome data were aligned with the known mouse genome sequence using HISAT2 software, and the reference genome was mm10 genome of mouse, and the selected parameters were default parameters. The HISAT2 software alignment results are shown in Table 2, and the matching rate of each sample transcriptome data and reference genome was more than 85%, which met the requirements of the next step analysis.

[0104] Table 2 Alignment results of each sample transcriptome data and mm10 genome

[0105]

[0106] Next, we used featureCounts software to annotate and quantitatively analyze the above data, and the GTF file used for annotation was the latest M23 version downloaded from Gencode website (download address: https: / / www.gencodegenes.org / mouse / ). The gene quantification analysis results after alignment and annotation are shown in Table 3.

[0107] Table 3. The data of each sample successfully annotated and quantified by featureCounts

[0108]

[0109] Then, we used DESeq2 software to analyze the gene expression data of each sample obtained above, using the parameters as the default parameters. Cluster analysis of 4 samples found that the data between Control and U0126 treatment had obvious differences, and at the same time showed that there was no cross contamination between samples Figure 11 ).

[0110] Next, the screening conditions of differential genes were set to 2-fold or more differential fold, p<0.05. Compared with the control group, there were 438 genes up-regulated in the U0126 group (Fold change>2, p<0.05); there were 434 genes down-regulated in the U0126 group (Fold change<-2, p <0.05). We selected part of the data to draw the differential gene expression heat map, as Figure 12 shown, the results showed that the reproducibility of these differential genes in Control and U0126 groups was good.

[0111] In order to analyze the function of these differential genes, it is necessary to perform enrichment analysis on the functional annotation of Gene Ontology of differential genes, and explore the biological significance of differential genes changed after U0126 treatment. The functional annotation of Gene Ontology is as Figure 13 shown, it can be found in the figure that the up-regulated and down-regulated genes are largely enriched in circulatory system development, blood vessel morphogenesis, nervous system development, anatomical structure morphogenesis, anatomical structure development and tissue morphogenesis, etc. The results show that U0126 is closely related to the development process of mouse cardiovascular system.

[0112] Figure 13 In the middle, 438 genes up-regulated after U0126 treatment and 434 genes down-regulated after U0126 treatment were clustered and dotplot was drawn.

[0113] Among the upregulated genes, Heart And Neural Crest Derivatives Expressed 2 ( Hand2 ) is a transcription factor that has been shown to induce fibroblast reprogramming into cardiac pacemakers, and Bmp4 These are growth factors that have been proven to be closely related to pacemaker cell development. Among the downregulated genes, erythropoietin receptor (Epor) knockout leads to epicardial detachment and ventricular hypoplasia in mice at E12-E13. Collagen 14A1 (Collagen type XIV alpha 1 chain, Col14a1 This gene is more frequently expressed in mice after E17.5 and in adult mice, and is associated with coronary artery disease. To further validate the results, we harvested EB differentiated to day 11 and processed them using real-time sequencing. q PCR technology was used to verify the expression of these genes. For example... Figure 14 As shown, the selected differentially expressed genes are consistent with the trends in transcriptome sequencing results.

[0114] in conclusion

[0115] The above results indicate that U0126 can effectively promote the differentiation of iPS cells into Shox2+ subtype pacemakers. Shox2+ cells induced by U0126 and RA differentiated... + Subtype pacemaker cells have similarities to Shox2 in vivo. + Cells share similar electrophysiological properties. U0126 may induce iPS cells to differentiate into pacemaker cells by activating the expression of Hand2 and Bmp4. This study established a method for effectively isolating Shox2. + and Shox2 + / Nkx2.5 + The model of two subtypes of pacemakers can be used to differentiate pacemakers with more uniform biological and electrophysiological characteristics, providing a stem cell source for the future development of biological pacemakers and the repair of damaged sinoatrial nodes.

[0116] Please note to all technical personnel: Although the present invention has been described according to the specific embodiments above, the inventive concept of the present invention is not limited to this invention. Any modifications that utilize the inventive concept will be included within the scope of protection of this patent.

[0117] The above merely describes the preferred embodiments of the present application, and the protection scope of the present application is not limited to the above-described embodiments. Any technical solution falling within the concept of the present application shall fall within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application shall also be considered as falling within the protection scope of the present application.

Claims

1. A method for inducing pluripotent stem cells to differentiate into sinoatrial node pacemaker cells, characterized in that, Includes the following steps: (1) Isolation and culture of mouse embryonic fibroblasts; (2) STEMCCA lentiviral infection reagent reprogrammed mouse embryonic fibroblasts into mouse iPS cells; (3) Selection of iPS cell clone communities; (4) iPS cell resuscitation; (5) iPS cells differentiate to obtain embryoid bodies (EB); (6) Shox2 was obtained by inducing differentiation of embryoid EB with the small molecule compound U0126. + Shox2 can be obtained by differentiation induced by the small molecule compound Retinoic Acid. + / Nkx2.5 + For pacemaker cell subtypes, the working concentration of compound U0126 was 10 μM, and the working concentration of compound RA was 1 μM. The small molecule compounds U0126 and RA were added on the second day of EB differentiation. (7) Patch-clamp technique to detect the electrophysiological characteristics of differentiated pacemaker-like cells.

2. The method according to claim 1, characterized in that, The isolation and culture of mouse embryonic fibroblasts in step (1) includes the following steps: (a) Take E12.5 mouse embryos, transfer the mice into a sterile culture dish of pre-cooled DPBS, and remove the head, limbs, internal organs and fluorescent protein-labeled parts of the mice with ophthalmic scissors and ophthalmic forceps. (b) Transfer the remaining mouse embryonic tissue to a new DPBS culture dish and wash the tissue three times with DPBS until no visible blood color remains; (c) Mouse embryonic tissue was placed into centrifuge tubes, and 0.5 mL of 0.25% Trypsin-EDTA digestion solution was added to each centrifuge tube; (d) After repeatedly pipetting the tissue 20 times, place the centrifuge tube in a 37°C incubator for 3 min, add mouse embryonic fibroblast culture medium to stop digestion, centrifuge for 5 min, discard the supernatant, add 1 mL of mouse embryonic fibroblast culture medium to each centrifuge tube to resuspend the cell pellet, and seed it into a 12-well plate, place it in a 37°C, 5% CO2 incubator, and change the medium daily; (e) Obtain Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26R mT / mG In mice, cells expressing Nkx2.5 are labeled with GFP green fluorescence, while cells expressing Shox2 can be labeled with LacZ live cell dye. After obtaining the mouse heart, it is divided into two parts along the coronal section using ophthalmic scissors. Under a fluorescence stereomicroscope, Shox2 is then labeled using pointed forceps and a 32G needle. + Sinoatrial node head cells, Shox2 + / Nkx2.5 + Cells at the sinoatrial node junction were completely isolated and collected; they were digested into single cells using type II collagenase and then cultured; after the cells adhered, the electrophysiological characteristics of the cells were detected using whole-cell patch-clamp technique. (f) The heart of wild-type E12.5, E14.5, and E16.5 mice, as well as mice 2 days and 4 weeks old, was sectioned using a vibrating microtome, and the pacing characteristics of the cells were detected using patch-clamp technique.

3. The method according to claim 1, characterized in that, Step (2) of infecting mouse embryonic fibroblasts with the STEMCCA lentiviral reagent to reprogram them into mouse iPS cells includes the following steps: (a) Day 0: Add 2 mL of 0.1% gelatin solution to each well of a sterile 6-well plate and incubate at 37°C for at least 30 minutes. After removing the gelatin solution, spread a layer of 1 x 10⁻⁶ gelatin solution into each well of the 6-well plate. 5 indivual Nkx2.5 IRESCre / + ; Shox2 LacZ / + ; ROSA26R mT / mG Mouse embryonic fibroblasts were added to 3 mL of fresh culture medium and cultured overnight in a 37°C, 5% CO2 incubator. On Day 1, the old culture medium was discarded, and 3 mL of fresh mouse embryonic fibroblast culture medium was added. 1.5 μL of Polybrene transfection reagent was added to each well, and the plate was incubated in a 37°C, 5% CO2 incubator until ready to add lentivirus. (ii) Thaw one tube of Mouse STEMCCA Constitutive Polycistronic OKSM lentivirus at room temperature, and immediately place it on ice after thawing, then add the required volume of lentivirus; (iii) After adding the required volume of virus directly to the well containing attached mouse embryonic fibroblasts, gently shake the six-well plate in a cross shape to promote the lentivirus to evenly cover the mouse embryonic fibroblasts. Place the six-well plate in a 37°C, 5% CO2 incubator overnight. (iv) Day 2: Discard the old culture medium and replace it with a complete iPS cell culture medium containing 15% FBS and LIF; Day 3 - Day 15: Replace with fresh iPS cell culture medium every day.

4. The method according to claim 1, characterized in that, The specific steps for iPS cell differentiation into embryoid bodies in step (5) are as follows: (I) When the growth density of ES or iPS cells reaches 80%, digest them with 0.25% Trypsin-EDTA and agitate them into a single-cell suspension; (II) Seed the cells at a certain density in culture dishes pre-coated with 0.1% gelatin, and incubate at 37℃ with 5% CO2 for 30 min. Then, aspirate any unattached cells from the supernatant and incubate at 300 × 10⁻⁶. g Centrifuge for 5 min; (III) Resuspend the cells in embryoid differentiation medium and seed them into a round-bottomed Ultra Low Attachment 96-well plate for hanging drop culture. Add 1000 cells to each well and add 300 μL of culture medium. (IV) After 24 hours of culture, granular spherical objects can be observed under a microscope, which are embryoid bodies (EB).

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