A SAN-like cell and a method of differentiating pluripotent stem cells into SAN-like cells in vitro

Abstract

This invention provides a method for differentiating SAN-like cells from pluripotent stem cells in vitro. The method for differentiating SAN-like cells from pluripotent stem cells in vitro includes S1, providing a population of pluripotent stem cells; S2, performing a first contact culture on the pluripotent stem cell population to obtain mesodermal cells; S3, performing a second contact culture on the mesodermal cells to obtain pSHF cells; and S4, performing a third contact culture on the pSHF cells to obtain SAN-like cells. This invention solves at least one of the following problems in the prior art: the induction efficiency of sinoatrial node (SAN)-like cells from pluripotent stem cells is generally low, usually only 30%–50%; and the differentiation cycle is long, the operation steps are cumbersome, and a variety of small molecule additives are required. In addition, the differentiation products contain a large number of NKX2.5 positive atrial or ventricular myocytes, resulting in insufficient purity, which still requires further cell separation and purification.

Description

Technical Field

[0001] This invention relates to the technical field of biology and new medicine, and more specifically, to a SAN-like cell and a method for differentiating pluripotent stem cells into SAN-like cells in vitro. Background Technology

[0002] The sinoatrial node (SAN) cells are the natural pacemaker cells of the human heart. Their core function is to autonomously generate rhythmic electrical impulses, guiding the heart's normal rhythmic beating. They can generate 60 to 100 impulses per minute and transmit them to all parts of the heart, controlling the contraction and expansion of the myocardium. They are a key cell group for maintaining normal cardiac physiological function. When SAN cells malfunction, it can lead to sinus arrhythmia, causing symptoms such as tachycardia, bradycardia, or irregular heart rate. In severe cases, this can be life-threatening. Currently, the main clinical treatment for such conditions is the implantation of an electronic pacemaker.

[0003] With the rapid development of regenerative medicine and stem cell technology, the directed differentiation of human induced pluripotent stem cells (iPSCs) into sinoatrial node (SAN)-like cells and the construction of biological pacemakers based on these cells has become a research hotspot to replace traditional electronic pacemakers, providing a new direction for the treatment of sinoatrial node dysfunction.

[0004] However, the relevant technologies have at least one of the following problems: In the existing technologies, the induction efficiency of sinoatrial node (SAN)-like cells obtained by pluripotent stem cell directed differentiation is generally low, usually only 30%–50%; and the differentiation cycle is long, the operation steps are complicated, and a variety of small molecule additives are required. At the same time, the differentiation products are mixed with a large number of NKX2.5 positive atrial or ventricular myocytes, which are not pure enough and still require further cell separation and purification. Summary of the Invention

[0005] This invention solves at least one of the following problems: the induction efficiency of sinoatrial node (SAN)-like cells obtained by pluripotent stem cell directed differentiation is generally low, usually only 30%–50%; and the differentiation cycle is long, the operation steps are complicated, and a variety of small molecule additives are required. At the same time, the differentiation products are mixed with a large number of NKX2.5 positive atrial or ventricular myocytes, which are not pure enough and still require further cell separation and purification.

[0006] To address the above problems, this invention provides a method for differentiating pluripotent stem cells into SAN-like cells in vitro, the method comprising: S1. Provide a population of pluripotent stem cells; S2. The pluripotent stem cell population is subjected to first contact culture to obtain mesodermal cells; S3. Perform a second contact culture on the mesodermal cells to obtain pSHF cells; S4. Perform third-contact culture on pSHF cells to obtain SAN-like cells.

[0007] Compared with existing technologies, the technical effects achieved by this solution are as follows: This solution obtains mesodermal cells through a first contact culture of pluripotent stem cell populations, obtains pSHF cells through a second contact culture, and obtains SAN-like cells through a third contact culture. This targeted induction precisely regulates the cell differentiation pathway, improves the induction efficiency of pluripotent stem cells to SAN-like cells, overcomes the problem of low differentiation efficiency in existing technologies, and shortens the differentiation cycle and simplifies the operation steps through a clear staged culture process. It also reduces the types and amounts of exogenous small molecule additives used and lowers the complexity of the induction system.

[0008] In one embodiment of the present invention, S2 includes: A population of pluripotent stem cells was contacted with a first culture medium containing Wnt signaling activator to induce mesodermal cell formation.

[0009] Compared with existing technologies, the technical effects achieved by this solution are as follows: This solution induces the formation of mesodermal cells by contacting a population of pluripotent stem cells with a first culture medium containing Wnt signaling activators, thereby precisely initiating and regulating the early key stages of pluripotent stem cell differentiation into the cardiac lineage, improving the induction efficiency and uniformity of mesodermal cells, and reducing the generation of non-target germ layer cells.

[0010] In one embodiment of the present invention, the concentration of the Wnt signal activator is 6µM-10µM; The Wnt signaling activator is selected from any one of the following: GSK3 inhibitor, CHIR99021, and GSK3-β inhibitor; The culture time of the and / or pluripotent stem cell population in the first culture medium is 40h to 50h.

[0011] Compared with existing technologies, the technical effects achieved by this solution are as follows: This solution uses any one of GSK3 inhibitors, CHIR99021, and GSK3-β inhibitors as Wnt signaling activators, controls the concentration of the Wnt signaling activator to 6µM-10µM, and culturees it in the first culture medium for 40-50 hours. This can stably and efficiently activate the Wnt signaling pathway, ensuring efficient induction of pluripotent stem cells to differentiate into mesodermal cells, while avoiding problems such as disordered cell differentiation and an increase in non-target lineage cells due to excessively strong signals or excessively long treatment times. At the same time, culturing for 40-50 hours can make the induction of mesodermal cells more uniform and the maturity appropriate, thereby improving the stability and controllability of the differentiation initiation stage and reducing cell heterogeneity.

[0012] In one embodiment of the present invention, S3 includes: Mesodermal cells were contacted with a second culture medium containing an FGF inhibitor; Mesodermal cells whose FGF inhibition had ended were contacted with a third culture medium containing a Wnt signaling inhibitor to obtain pSHF cells.

[0013] Compared with existing technologies, the technical effects achieved by this approach are as follows: This approach first treats mesodermal cells with a culture medium containing an FGF inhibitor. After the FGF inhibition ends, pSHF cells are induced by a culture medium containing a Wnt signaling inhibitor. This enables precise temporal regulation of two key signaling pathways, avoiding signal interference and cytotoxicity caused by the simultaneous action of two inhibitors. While precisely blocking FHF specialization and guiding cells to develop into the pSHF lineage, it also more gently and controllably inhibits Wnt signaling to promote the targeted specialization of cardiac pSHF cells, reducing premature differentiation and the generation of hybrid cells in the atrial and ventricular myocardial lineages, and further improving the uniformity and differentiation stability of cardiac progenitor cells.

[0014] In one embodiment of the present invention, S4 includes: pSHF cells were contacted with a fourth culture medium containing TGF-β inhibitors, FGF inhibitors, and RA, and after culture, they were further differentiated to obtain SAN-like cells.

[0015] Compared with existing technologies, the technical effects achieved by this solution are as follows: In step S4, this solution contacts pSHF cells with a fourth culture medium containing TGF-β inhibitors, FGF inhibitors, and retinoic acid (RA). This allows for synergistic regulation of signaling pathways at the critical stage of cardiac progenitor cells specializing into the sinoatrial node lineage, precisely driving the cells to differentiate into SAN cells. This effectively inhibits the differentiation fate of non-pacing cardiomyocytes such as atrial and ventricular myocardium, thereby reducing the production of NKX2.5 positive contaminants, improving the specificity and purity of SAN-like cell specialization, and optimizing the differentiation process and increasing induction efficiency.

[0016] In one embodiment of the present invention, in S3, the concentration of the FGF inhibitor is 50µM-200µM; In S4, the concentration of TGF-β inhibitor was 1µM-2µM, the concentration of FGF inhibitor was 0.5µM-1µM, and the concentration of RA was 0.25µM-0.5µM. The FGF inhibitor is selected from at least one of PD173074, SU5402 and BGJ398; And / or TGF-β inhibitor A8301; Among them, FGF inhibitors are used to inhibit the specialization of FHF.

[0017] Compared with the prior art, the technical effects achieved by adopting this technical solution are as follows: This solution selects at least one of PD173074, SU5402, and BGJ398 as FGF inhibitors, and selects A8301 as TGF-β inhibitors. In S3, the concentration of FGF inhibitor is controlled at 50µM-200µM, and in S4, the concentration of TGF-β inhibitor is controlled at 1µM-2µM, the concentration of FGF inhibitor is controlled at 0.5µM-1µM, and the concentration of RA is controlled at 0.25µM-0.5µM. Among these, FGF inhibitors gently regulate cell proliferation and differentiation, promoting the specific formation of cardiac lineage precursor cells, inhibiting premature differentiation of non-myocardial lineage and atrial and ventricular myocytes, reducing the production of NKX2.5-positive contaminating cells, and improving the homogeneity and purity of cardiac precursor cells. Furthermore, FGF inhibitors specifically inhibit the specialization of the first cardiac region (FHF), promoting the development of mesodermal cells towards the posterior second cardiac region (pSHF) lineage while maintaining cell viability. Simultaneously, TGF-β inhibitors synergistically optimize the signaling microenvironment, further enhancing the directional specialization efficiency and product purity of sinoatrial node-like cells, improving the stability and reproducibility of the differentiation system, and avoiding the problems of high differentiation heterogeneity and low pacemaker cell proportion caused by signaling pathway disorders.

[0018] In one embodiment of the present invention, the concentration of the Wnt signal inhibitor is 8µM-10µM; the Wnt signal inhibitor is selected from at least one of IWP-2, IWR-1 and XAV939.

[0019] Compared with existing technologies, the technical effects achieved by this solution are as follows: This solution selects at least one of IWP-2, IWR-1, and XAV939 as a Wnt signaling inhibitor, and controls the concentration of the Wnt signaling inhibitor to be 8µM-10µM. This allows for the specific and gentle inhibition of the Wnt signaling pathway during the differentiation stage of cardiac progenitor cells, avoiding cell differentiation disorder caused by excessive signal activation or uneven inhibition, effectively blocking the differentiation of non-target myocardial lineages, and improving the accuracy and uniformity of sinoatrial node progenitor cell directional induction.

[0020] In one embodiment of the present invention, S1 includes: Human pluripotent stem cell lines were cultured in mTeSR™ medium and passaged to obtain a pluripotent stem cell population. Among them, human pluripotent stem cell lines were passaged at a ratio of 1:60 and cultured for 3-4 days; or Human pluripotent stem cell lines were cultured in mTeSR™ medium and differentiated to obtain a pluripotent stem cell population. Human pluripotent stem cell lines were differentiated at a ratio of 1:10 and cultured for 3-4 days.

[0021] Compared with existing technologies, the technical effects achieved by this solution are as follows: In step S1, human pluripotent stem cell lines are cultured in mTeSR™ medium and passaged at a ratio of 1:60 for 3-4 days to obtain a uniform pluripotent stem cell population, or plated at a ratio of 1:10 for 3-4 days for direct differentiation. This ensures that stem cells are in a good pluripotent state, with uniform cell density and stable growth at the initiation stage of differentiation, avoiding problems such as asynchronous differentiation initiation and high cell heterogeneity caused by excessively high or low cell density. At the same time, the use of mTeSR™ medium can maintain the undifferentiated state of stem cells and reduce spontaneous differentiation, thereby improving the controllability and reproducibility of the overall differentiation process.

[0022] In one embodiment of the present invention, the human pluripotent stem cell line is selected from any one of H7, HN4, and induced pluripotent stem cell lines.

[0023] Compared with existing technologies, the technical effects achieved by this solution are as follows: This solution selects any one of H7, HN4, and induced pluripotent stem cell lines as the source of human pluripotent stem cells. H7, HN4, and induced pluripotent stem cell lines all possess stable pluripotency and good cardiac lineage differentiation potential, with strong compatibility and consistent culture characteristics. This ensures that pSHF cell differentiation and subsequent sinoatrial node-like cell specialization can be efficiently initiated under the same induction system, thereby improving the universality and reproducibility of the differentiation method and further ensuring the purity and functional stability of the final obtained SAN-like cells.

[0024] On the other hand, the present invention also provides a SAN-like cell, which is prepared by the method of differentiating pluripotent stem cells into SAN-like cells in vitro in any of the above examples; SAN-like cells include at least one SAN-specific gene among TBX18, SHOX4, and HCN4.

[0025] Compared with existing technologies, the technical effects achieved by adopting this technical solution are as follows: This solution can achieve the technical effects corresponding to any of the above examples, which will not be elaborated here.

[0026] By adopting the technical solution of the present invention, the following technical effects can be achieved: This invention improves the efficiency of directional differentiation of pluripotent stem cells into SAN-like cells by sequentially performing a first contact culture to obtain mesodermal cells, a second contact culture to obtain pSHF cells, and a third contact culture to obtain SAN-like cells. This direct induction precisely regulates the cell differentiation pathway, overcomes the problem of low differentiation efficiency in existing technologies, and shortens the differentiation cycle and simplifies the operation steps through a clear staged culture process. It also reduces the types and amounts of exogenous small molecule additives used and lowers the complexity of the induction system. Simultaneously, in step S4, pSHF cells are contacted with a fourth culture medium containing TGF-β inhibitors, FGF inhibitors, and retinoic acid (RA). This allows for synergistic regulation of signaling pathways at the critical stage of cardiac progenitor cells' specialization into the sinoatrial node lineage, precisely driving the cells to differentiate into SAN cells. This effectively inhibits the differentiation fate of non-pacing cardiomyocytes such as atrial and ventricular myocardium, thereby reducing the production of NKX2.5 positive contaminants, improving the specificity and purity of SAN-like cell specialization, and optimizing the differentiation process and increasing induction efficiency. Attached Figure Description

[0027] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings to be used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Figure 1 A flowchart of a method for differentiating pluripotent stem cells into SAN-like cells in vitro, provided as an embodiment of the present invention; Figure 2 These are immunofluorescence images of cardiomyocytes on day 20 of Example 1, Control Group 1, and Control Group 2; Figure 3 Immunofluorescence images of HCN4 and TBX18 expression in differentiated cells of Example 1; Figure 4 This is a comparison chart of the differentiation efficiency of SAN-like cells in Example 1 and Control Group 2; Figure 5 The results are quantitative PCR analysis of pluripotency genes and SAN-related genes in SAN cells differentiated from Example 1 and Control Group 2. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0029] like Figure 1As shown, this invention provides a method for differentiating pluripotent stem cells into SAN-like cells in vitro, the method comprising: S1. Provide a population of pluripotent stem cells; S2. The pluripotent stem cell population is subjected to first contact culture to obtain mesodermal cells; S3. Perform a second contact culture on the mesodermal cells to obtain pSHF cells; S4. Perform third-contact culture on pSHF cells to obtain SAN-like cells.

[0030] Understandably, this approach improves the efficiency of pluripotent stem cell differentiation into SAN-like cells by sequentially performing first-contact culture to obtain mesodermal cells, second-contact culture to obtain pSHF cells, and third-contact culture to obtain SAN-like cells. This targeted induction precisely regulates the cell differentiation pathway, overcoming the problem of low differentiation efficiency in existing technologies. At the same time, the clearly defined staged culture process shortens the differentiation cycle, simplifies the operation steps, and reduces the types and amounts of exogenous small molecule additives used, thereby reducing the complexity of the induction system.

[0031] In some embodiments provided by the present invention, S2 includes: contacting a population of pluripotent stem cells with a first culture medium containing a Wnt signaling activator to induce mesodermal cell formation.

[0032] Understandably, this approach aims to precisely initiate and regulate the early critical stages of pluripotent stem cell differentiation into the cardiac lineage by contacting a pluripotent stem cell population with a first culture medium containing Wnt signaling activators to induce mesodermal cell formation. This improves the induction efficiency and uniformity of mesodermal cells and reduces the generation of non-target germ layer cells.

[0033] In some embodiments provided by the present invention, the concentration of the Wnt signaling activator is 6µM-10µM; the Wnt signaling activator is selected from any one of GSK3 inhibitors, CHIR99021, and GSK3-β inhibitors; and / or the pluripotent stem cell population is cultured in the first culture medium for 40h-50h.

[0034] Understandably, this protocol selects any one of the GSK3 inhibitors, CHIR99021, and GSK3-β inhibitors as Wnt signaling activators, controls the concentration of the Wnt signaling activator to 6µM-10µM, and culturees in the first culture medium for 40-50 hours. This can stably and efficiently activate the Wnt signaling pathway, ensuring efficient induction of pluripotent stem cells to differentiate into mesodermal cells, while avoiding problems such as disordered cell differentiation and an increase in non-target lineage cells due to excessively strong signals or excessively long treatment times. At the same time, culturing for 40-50 hours can make the induction of mesodermal cells more uniform and the maturity appropriate, thereby improving the stability and controllability of the differentiation initiation stage and reducing cell heterogeneity.

[0035] In some embodiments provided by the present invention, S3 includes: contacting mesodermal cells with a second culture medium containing an FGF inhibitor; and contacting the mesodermal cells after FGF inhibition has ended with a third culture medium containing a Wnt signaling inhibitor to obtain pSHF cells.

[0036] Understandably, this protocol first treats mesodermal cells with a medium containing an FGF inhibitor. After the FGF inhibition ends, pSHF cells are induced by a medium containing a Wnt signaling inhibitor. This allows for precise, time-sequential regulation of two key signaling pathways, avoiding signal interference and cytotoxicity caused by the simultaneous action of the two inhibitors. While precisely blocking FHF specialization and guiding cells to develop into the pSHF lineage, it also more gently and controllably inhibits Wnt signaling to promote the targeted specialization of cardiac pSHF cells. This reduces premature differentiation and the generation of hybrid cells in the atrial and ventricular myocardial lineages, further enhancing the uniformity and differentiation stability of cardiac progenitor cells.

[0037] In some embodiments provided by the present invention, S4 includes: contacting pSHF cells with a fourth culture medium containing a TGF-β inhibitor, an FGF inhibitor, and RA to obtain SAN-like cells.

[0038] Understandably, in step S4 of this protocol, contacting pSHF cells with a fourth culture medium containing TGF-β inhibitors, FGF inhibitors, and retinoic acid (RA) can synergistically regulate signaling pathways at the critical stage of cardiac progenitor cells' specialization into the sinoatrial node lineage. This precisely drives the cells to differentiate into SAN cells, effectively inhibiting the differentiation fate of non-pacing cardiomyocytes such as atrial and ventricular myocardium. Consequently, it reduces the production of NKX2.5-positive contaminating cells, improves the specificity and purity of SAN-like cell specialization, and optimizes the differentiation process and increases induction efficiency.

[0039] In some embodiments provided by the present invention, in S3, the concentration of the FGF inhibitor is 50µM-200µM; in S4, the concentration of the TGF-β inhibitor is 1µM-2µM, the concentration of the FGF inhibitor is 0.5µM-1µM, and the concentration of RA is 0.25µM-0.5µM; the FGF inhibitor is selected from at least one of PD173074, SU5402, and BGJ398; and / or the TGF-β inhibitor is selected from A8301; wherein, the FGF inhibitor is used to inhibit the specialization of FHF.

[0040] Understandably, this protocol selects at least one of PD173074, SU5402, and BGJ398 as an FGF inhibitor and A8301 as a TGF-β inhibitor. In S3, the concentration of the FGF inhibitor is controlled at 50µM-200µM, and in S4, the concentration of the TGF-β inhibitor is controlled at 1µM-2µM, the concentration of the FGF inhibitor at 0.5µM-1µM, and the concentration of RA at 0.25µM-0.5µM. The FGF inhibitor gently regulates cell proliferation and differentiation, promotes the specific formation of cardiac lineage precursor cells, inhibits premature differentiation of non-cardiac lineage and atrial and ventricular myocytes, reduces the production of NKX2.5 positive contaminants, and improves the homogeneity and purity of cardiac precursor cells. Furthermore, the FGF inhibitor specifically inhibits the specialization of the first cardiac region (FHF), causing mesodermal cells to develop towards the posterior second cardiac region (pSHF) lineage while maintaining cell viability. Meanwhile, TGF-β inhibitors synergistically optimize the signal microenvironment, further enhancing the directional specialization efficiency and product purity of sinoatrial node-like cells, improving the stability and reproducibility of the differentiation system, and avoiding the problems of high differentiation heterogeneity and low pacemaker cell ratio caused by signal pathway disorders.

[0041] In some embodiments provided by the present invention, the concentration of the Wnt signaling inhibitor is 8µM-10µM; the Wnt signaling inhibitor is selected from at least one of IWP-2, IWR-1 and XAV939.

[0042] Understandably, this protocol selects at least one of IWP-2, IWR-1, and XAV939 as a Wnt signaling inhibitor, and controls the concentration of the Wnt signaling inhibitor to be 8µM-10µM. This allows for the specific and gentle inhibition of the Wnt signaling pathway during the differentiation stage of cardiac progenitor cells, avoiding cell differentiation disorder caused by excessive signal activation or uneven inhibition, effectively blocking the differentiation of non-target myocardial lineages, and improving the accuracy and uniformity of sinoatrial node progenitor cell directional induction.

[0043] In some embodiments provided by the present invention, S1 includes: Human pluripotent stem cell lines were cultured in mTeSR™ medium and passaged to obtain a pluripotent stem cell population; the human pluripotent stem cell lines were passaged at a ratio of 1:60 and cultured for 3-4 days. or Human pluripotent stem cell lines were cultured in mTeSR™ medium to differentiate them and obtain a pluripotent stem cell population; the human pluripotent stem cell lines were differentiated at a ratio of 1:10 and cultured for 3-4 days.

[0044] Understandably, in step S1 of this protocol, human pluripotent stem cell lines are cultured in mTeSR™ medium and passaged at a ratio of 1:60 for 3-4 days to obtain a uniform pluripotent stem cell population, or plated at a ratio of 1:10 for 3-4 days for direct differentiation. This ensures that stem cells are in a good pluripotent state, with uniform cell density and stable growth at the initiation stage of differentiation, avoiding problems such as asynchronous differentiation initiation and high cell heterogeneity caused by excessively high or low cell density. At the same time, the use of mTeSR™ medium can maintain the undifferentiated state of stem cells and reduce spontaneous differentiation, thereby improving the controllability and reproducibility of the overall differentiation process.

[0045] In some embodiments provided by the present invention, the human pluripotent stem cell line is selected from any one of H7, HN4, and induced pluripotent stem cell lines.

[0046] Understandably, this protocol selects any one of H7, HN4, and induced pluripotent stem cell lines as the source of human pluripotent stem cells. H7, HN4, and induced pluripotent stem cell lines all possess stable pluripotency and good cardiac lineage differentiation potential, with strong compatibility and consistent culture characteristics. This ensures that pSHF cell differentiation and subsequent sinoatrial node-like cell specialization can be efficiently initiated under the same induction system, thereby improving the universality and reproducibility of the differentiation method and further ensuring the purity and functional stability of the final obtained SAN-like cells.

[0047] A SAN-like cell, which is prepared by the method of differentiating pluripotent stem cells into SAN-like cells in vitro in any of the above examples; SAN-like cells include at least one SAN-specific gene among TBX18, SHOX4, and HCN4.

[0048]

Example 1

[0049] It should be noted that the ReLeSR™ mentioned in the embodiments of the present invention is a recombinant enzymatic hydrolysis passage reagent for human pluripotent stem cells, and the mTeSR™ culture medium is a modified embryonic stem cell-specific culture medium.

[0050] S2: First contact culture (mesoderm cell induction) The pluripotent stem cell population obtained in S1 was subjected to first contact culture in the first culture medium for 48 hours to induce mesodermal cell formation. The first culture medium was RPMI 1640 medium supplemented with 6 µM CHIR99021 and insulin-free B27 as an additive. After the culture was completed, the medium was replaced with basal medium containing RPMI 1640 and insulin-free B27 and cultured for 24 hours.

[0051] S3: Second contact culture (pSHF specialization induction) On day 2 of differentiation, the mesodermal cells obtained in S2 were subjected to a second contact culture in the second medium for 48 hours. The second medium was RPMI 1640 medium supplemented with 50 nM PD173074 and insulin-free B27 as an additive. PD173074 was at a low concentration (subcytotoxic concentration), which could inhibit the specialization of the first heart zone (FHF) while maintaining cell viability and promoting the development of mesodermal cells towards the posterior second heart zone (pSHF) lineage. In the following 48 to 96 hours, the cells were cultured in a third medium to obtain pSHF cells. The third medium was RPMI 1640 medium supplemented with 8 µM XAV939 and insulin-free B27 as an additive.

[0052] S4: Third contact culture (SAN cell specialization) After Wnt inhibition, pSHF cells obtained in S3 were subjected to third-contact culture in the fourth medium for 72 hours. After culture, they were transferred to RPMI 1640 medium (with insulin B27 additive, insulin concentration 3 µg / mL) and continued to differentiate until day 20. During differentiation, metabolic screening was performed starting on day 15. Cells were cultured in glucose-free RPMI 1640 medium supplemented with lactate only to remove free non-muscle cells, ultimately obtaining high-purity SAN-like cells. The fourth medium was RPMI 1640 medium supplemented with 1 µM A8301, 0.5 µM PD173074, and 0.25 µM retinoic acid (RA), with insulin B27 added as an additive to synergistically regulate signaling pathways, drive SAN cell specialization, promote the upregulation of SAN-specific markers ISL1 and SHOX2 expression, and actively inhibit NKX2.5 expression.

[0053]

Example 2

[0054] S2: First contact culture (mesoderm cell induction) The pluripotent stem cell population obtained in S1 was subjected to first-contact culture in the first culture medium for 50 hours to induce mesodermal cell formation. The first culture medium was RPMI 1640 medium supplemented with 10 µM CHIR99021 and insulin-free B27 additive. After the culture was completed, the medium was replaced with basal medium containing RPMI 1640 and insulin-free B27 and cultured for 24 hours.

[0055] S3: Second contact culture (pSHF specialization induction) On day 2 of differentiation, the mesodermal cells obtained from S2 were subjected to a second contact culture in a second medium for 48 hours. The second medium was RPMI 1640 medium supplemented with 200 nM SU5402, with insulin-free B27 added as an additive. SU5402 was at a low concentration (subcytotoxic concentration), which could inhibit the specialization of the first heart zone (FHF) while maintaining cell viability and promoting the development of mesodermal cells towards the posterior second heart zone (pSHF) lineage. In the following 48 to 96 hours, the cells were cultured in a third medium to obtain pSHF cells. The third medium was RPMI 1640 medium supplemented with 10 µM IWP-2, with insulin-free B27 added as an additive.

[0056] S4: Third contact culture (SAN cell specialization) After Wnt inhibition, pSHF cells obtained in S3 were subjected to third-contact culture in the fourth medium for 72 hours. After culture, they were transferred to RPMI 1640 medium (with insulin B27 additive, insulin concentration of 8 µg / mL) and continued to differentiate until day 20. During differentiation, metabolic screening was performed starting on day 15. Cells were cultured in glucose-free RPMI 1640 medium supplemented with lactate only to remove free non-muscle cells, ultimately obtaining high-purity SAN-like cells. The fourth medium was RPMI 1640 medium supplemented with 2 µM A8301, 1 µM SU5402 and 0.5 µM retinoic acid (RA), with insulin B27 added as an additive to synergistically regulate signaling pathways, drive SAN cell specialization, promote the upregulation of SAN-specific markers such as ISL1 and SHOX2, and actively inhibit NKX2.5 expression.

[0057]

Example 3

[0058] S2: First contact culture (mesoderm cell induction) The pluripotent stem cell population obtained in S1 was subjected to first-contact culture in the first culture medium for 45 hours to induce mesodermal cell formation. The first culture medium was RPMI 1640 medium supplemented with 8 µM CHIR99021 and insulin-free B27 additive. After the culture was completed, the medium was replaced with basal medium containing RPMI 1640 and insulin-free B27 and cultured for 24 hours.

[0059] S3: Second contact culture (pSHF specialization induction) On day 2 of differentiation, the mesodermal cells obtained from S2 were subjected to a second contact culture in a second medium for 48 hours. The second medium was RPMI 1640 medium supplemented with 100 nM BGJ398, with insulin-free B27 added as an additive. BGJ398 was at a low concentration (subcytotoxic concentration), which could inhibit the specialization of the first heart zone (FHF) while maintaining cell viability and promoting the development of mesodermal cells towards the posterior second heart zone (pSHF) lineage. In the following 48 to 96 hours, the cells were cultured in a third medium to obtain pSHF cells. The third medium was RPMI 1640 medium supplemented with 9 µM IWR-1, with insulin-free B27 added as an additive.

[0060] S4: Third contact culture (SAN cell specialization) After Wnt inhibition, pSHF cells obtained in S3 were subjected to third-contact culture in the fourth medium for 72 hours. After culture, they were transferred to RPMI 1640 medium (with insulin B27 additive, insulin concentration of 5 µg / mL) and continued to differentiate until day 20. During differentiation, metabolic screening was performed starting on day 15. Cells were cultured in glucose-free RPMI 1640 medium supplemented with lactate only to remove free non-muscle cells, ultimately obtaining high-purity SAN-like cells. The fourth medium was RPMI 1640 medium supplemented with 1.5 µM A8301, 1 µM BGJ398, and 0.3 µM retinoic acid (RA), with insulin B27 added as an additive to synergistically regulate signaling pathways, drive SAN cell specialization, promote the upregulation of SAN-specific markers such as ISL1 and SHOX2, and actively inhibit NKX2.5 expression.

[0061]

Control Group 1

[0062]

Control Group 2

[0063] Tables 1-3 show the SAN cell differentiation timelines for Example 1, Control Group 1, and Control Group 2.

[0064] Table 1. Timeline of SAN cell differentiation (Example 1) Table 2. SAN cell differentiation timeline (control group 1) Table 3. SAN cell differentiation timeline (control group 2) Abbreviations in Tables 1-3: CHIR: CHIR99021; PD: PD173074; XAV: XAV939; RA: Retinic acid; Ins: Insulin; Lactate: Lactic acid; Comparative analysis of experimental data like Figure 2 The image shows immunofluorescence images of cardiomyocytes on day 20 of Example 1, Control Group 1, and Control Group 2, demonstrating the expression of the ventricular marker NKX2.5 on day 20 of differentiation. On day 20, Control Group 2 produced a high proportion of NKX2.5+ cells in its cardiomyocytes, while Control Group 1 and Example 1 showed significantly reduced NKX2.5 expression in their cardiomyocytes on day 20, indicating successful specialization towards a sinoatrial node (SAN)-like fate.

[0065] like Figure 3 The image shown is an immunofluorescence image of HCN4 and TBX18 expression in differentiated cells from Example 1. It illustrates the co-expression of the key pacemaker cell-specific markers HCN4 and TBX18 with αSA in SAN-like cells obtained through Example 1 on day 20. Figure 3 Strong common expression in China, plus Figure 2 The confirmed NKX2.5 negative phenotype demonstrates the successful and efficient generation of SAN-like cells.

[0066] like Figure 4As shown, this illustrates the differentiation efficiency of SAN-like cells in Example 1. In Figure (A), quantitative analysis using immunofluorescence staining shows that the proportion of NKX2.5+ cells in Example 1 was significantly reduced compared to control group 2. In Figure (B), analysis of spontaneous beat frequency indicates that the cells derived in Example 1 exhibited a faster and more sustained pacemaker-like rhythm. In Figure (C), representative action potential recordings confirm that the cells guided by Example 1 displayed typical sinoatrial node electrophysiological characteristics, which differed from the ventricular-like action potentials observed in control group 2.

[0067] like Figure 5 As shown, this is a quantitative PCR analysis of pluripotency genes and SAN-related genes in SAN cells differentiated in Example 1. Gene expression profiling analysis by qPCR showed that the cells successfully transitioned from a pluripotent state to a targeted cardiac pacemaker cell fate. Compared with undifferentiated embryonic stem cells (ESCs) and ventricular-like cardiomyocytes generated from control group 2, the cells differentiated in Example 1 showed downregulation of pluripotency markers (comparable to control group 2), while the expression of specific SAN-related genes (e.g., SHOX2, HCN4, TBX18) was significantly upregulated.

[0068] It should be noted that, in the embodiments of this invention, the following are mentioned: FGF inhibitor is a fibroblast growth factor inhibitor; TGF-β inhibitor is a transforming growth factor-β inhibitor; CHIR99021 is a Wnt signaling activator, namely a glyceraldehyde-3-triphosphate dehydrogenase inhibitor, whose chemical name is 6-[[2-[[4-(2,4-dichlorophenyl)-5-pyrimidinyl]amino]ethyl]amino]nicotinic acid; XAV939 is a Wnt signaling inhibitor, whose chemical name is 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one; IWP-2 is The Wnt signaling inhibitor has the chemical name: N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthiophene[3,2-d]pyrimidin-2-yl)thio]acetamide; IWR-1 is a Wnt signaling inhibitor with the chemical name: 4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methylbridge-2H-isoindol-2-yl]-N-8-quinolinylbenzamide; PD173074 is an FGF inhibitor, i.e., a fibroblast growth factor receptor inhibitor with the chemical name: 1 -tert-butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]urea; SU5402 is an FGF inhibitor, its chemical name is: 2-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-4-methyl-1H-pyrrole-3-propionic acid; BGJ398 is an FGF inhibitor, its chemical name is: 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-[6-[[4-(4-methylpiperazin-1-yl)phenyl]amino]pyrimidin-4-yl]-1-methylurea; A8301 is T GF-β inhibitor, also known as transforming growth factor-β inhibitor, has the chemical name 3-(6-methyl-2-pyridyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-thiocarboxamide; GSK3 is glycogen synthesis kinase 3; ISL1 is insulin gene-enhancing binding protein 1; SHOX2 is dwarf homeobox gene 2; NKX2.5 is homeobox transcription factor; HCN4 is; TBX18 is T-box transcription factor 18; SHOX4 is dwarf homeobox gene 4; mTeSR™ medium is a modified embryonic stem cell-defined medium; ReLeSR™ is a recombinant enzymatic digestion and passage reagent for human pluripotent stem cells.

[0069] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A method for differentiating pluripotent stem cells into SAN-like cells in vitro, characterized in that, The method includes: S1. Provide a population of pluripotent stem cells; S2. The pluripotent stem cell population is subjected to first contact culture to obtain mesodermal cells; S3. The mesodermal cells are subjected to a second contact culture to obtain pSHF cells; S4. The pSHF cells are subjected to third-contact culture to obtain SAN-like cells.

2. The method according to claim 1, characterized in that, S2 includes: The pluripotent stem cell population was contacted with a first culture medium containing a Wnt signaling activator to induce the formation of mesodermal cells.

3. The method according to claim 2, characterized in that, The concentration of the Wnt signal activator is 6µM-10µM; And / or the Wnt signaling activator is selected from any one of GSK3 inhibitors, CHIR99021, and GSK3-β inhibitors; And / or the pluripotent stem cell population is cultured in the first culture medium for 40h to 50h.

4. The method according to claim 1, characterized in that, S3 includes: The mesodermal cells were contacted with a second culture medium containing an FGF inhibitor; The mesodermal cells, after FGF inhibition has ended, are contacted with a third culture medium containing a Wnt signaling inhibitor to obtain the pSHF cells.

5. The method according to claim 4, characterized in that, S4 includes: The pSHF cells were contacted with a fourth culture medium containing a TGF-β inhibitor, the FGF inhibitor, and RA, and after culture, they were further differentiated to obtain the SAN-like cells.

6. The method according to claim 5, characterized in that, In S3, the concentration of the FGF inhibitor is 50µM-200µM; In S4, the concentration of the TGF-β inhibitor is 1µM-2µM, the concentration of the FGF inhibitor is 0.5µM-1µM, and the concentration of the RA is 0.25µM-0.5µM. And / or the FGF inhibitor is selected from at least one of PD173074, SU5402 and BGJ398; And / or the TGF-β inhibitor is selected as A8301; The FGF inhibitor is used to inhibit the specialization of FHF.

7. The method according to claim 5, characterized in that, The concentration of the Wnt signaling inhibitor is 8µM-10µM; And / or the Wnt signaling inhibitor is selected from at least one of IWP-2, IWR-1 and XAV939.

8. The method according to claim 1, characterized in that, S1 includes: Human pluripotent stem cell lines were cultured in mTeSR™ medium and passaged to obtain the pluripotent stem cell population. The human pluripotent stem cell line was passaged at a ratio of 1:60 and cultured for 3-4 days. or Human pluripotent stem cell lines were cultured in mTeSR™ medium and differentiated to obtain the pluripotent stem cell population. The human pluripotent stem cell line was differentiated at a ratio of 1:10 and cultured for 3-4 days.

9. The method according to claim 8, characterized in that, The human pluripotent stem cell line is selected from any one of H7, HN4, and induced pluripotent stem cell lines.

10. A SAN-like cell, characterized in that, The SAN-like cells are prepared by the method of differentiating pluripotent stem cells into SAN-like cells in vitro as described in any one of claims 1-9; The SAN-like cells include at least one SAN-specific gene selected from TBX18, SHOX4, and HCN4.

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