Method suitable for differentiating atrial muscle cells by iPSC (induced pluripotent stem cells) from different sources

Abstract

The invention relates to a method for producing atrial muscle cells by differentiating iPSC (induced pluripotent stem cells). The method is suitable for differentiating the iPSC from different sources to obtain the atrial muscle cells with high conversion rate. The method mainly comprises the steps that RPMI 1640 + B27minus inslin + LAA is adopted as a differentiation basic solution, small molecules CHIR99021, Wnt-C59 and RA are used in cooperation, the survival number of cells in the atrial muscle cell differentiation process is effectively increased, meanwhile, the yield of the atrial muscle cells is increased, and powerful support is provided for industrial application of iPSC differentiation of the atrial muscle cells.

Description

Technical Field

[0001] The present invention relates to a method for differentiating induced pluripotent stem cells (iPSCs) into atrial myocytes (AMs), which is suitable for preparing atrial myocytes by differentiating iPSCs from different sources. Background Art

[0002] AMs differentiated from human iPSCs (iPSC-AMs) can be observed to beat spontaneously in culture, and the expression of action potentials and ion channels can be detected in vitro. By incorporating iPSC-AMs cells carrying specific gene mutations, the pathological characteristics of the disease can be reproduced in vitro, helping to gain a deeper understanding of the pathogenesis of the disease. iPSC-AMs can be used to simulate the electrophysiological characteristics of arrhythmias such as atrial fibrillation, providing an in vitro model for the development of related drugs. iPSC-AMs can also be used as a platform for drug screening to test the effects of new drugs on atrial myocyte function. In addition, atrial myocyte contractility is generally weaker than that of ventricular myocytes, but their contraction is crucial for maintaining normal cardiac pumping function. iPSC-AMs can be combined with matrigel, fibroblasts, endothelial cells, etc. to prepare 3D tissues such as myocardial sheets and myospheres for the treatment of atrial myocardial diseases.

[0003] At present, improving the efficiency of atrial myocyte differentiation from iPSCs is still a challenge, and further optimization of differentiation conditions is needed. At the same time, it is also urgent to develop a differentiation method for atrial myocytes that is suitable for different diseases, different healthy samples, and gene-edited iPSCs. Existing atrial myocyte differentiation methods mostly use RPMI 1640+human albumin+L-ascorbic acid as the differentiation base liquid, combined with small molecule additives CHIR99021, IWP-2, and growth factors such as Noggin, BMP4, ActivinA, and FGF2 [1]. Currently, most of the existing methods cause a large number of cell death in the early stages of differentiation, and the efficiency of cardiomyocyte differentiation is generally not high, most of which remain in the range of 50-70%, which greatly affects the possibility of industrial application of iPSC-AMs. In addition, these differentiation methods are currently mostly only applicable to the differentiation of atrial myocytes from iPSCs derived from healthy samples. For iPSCs derived from different diseases, or gene-edited iPSCs, there are still many difficulties to be overcome in differentiating usable atrial myocytes.

[0004] iPSC differentiation into atrial myocytes involves four main stages. First, iPSCs are induced to differentiate into mesoderm under stimulation with growth factors such as BMP4 and Activin A, or the small molecule CHIR99021. Then, Wnt signaling inhibitors are added to regulate the differentiation of mesoderm cells into cardiac progenitor cells, causing the cells to form multilayers or aggregates. Finally, atrial-specific growth factors or small molecule compounds are added to promote the specific differentiation of cardiac progenitor cells into atrial myocytes. The two most important aspects of iPSC differentiation into atrial myocytes are: first, the iPSC state must be adjusted to achieve typical iPSC morphology, stable proliferation, and a passage viability of over 90%, allowing for the initiation of atrial myocyte differentiation; second, maintaining high confluence throughout the differentiation process and ensuring the cells have the ability to proliferate continuously during the transformation process. Summary of the Invention

[0005] The overall efficiency of the currently reported methods for differentiating atrial myocytes from iPSCs is relatively low and cannot yet support industrial scale-up production, which has affected the development of clinical applications of iPSC-differentiated atrial myocytes. In addition, the current differentiation methods are mostly used for the differentiation of atrial myocytes from healthy iPSCs and are not universally applicable. The present invention mainly provides a method for differentiating atrial myocytes with high differentiation efficiency and strong universality (suitable for healthy iPSCs, disease-derived iPSCs, and gene-edited iPSCs).

[0006] For the above invention, the specific technical solution is:

[0007] This protocol utilizes continuous stimulation with low-concentration CHIR99021 to reduce the number of iPSCs that die and enhance their proliferation during the transition to mesoderm. Wnt-C59, an inhibitor of the Wnt signaling pathway, is then used to induce the transformation of mesoderm cells into cardiac progenitor cells. Later in the Wnt-C59 treatment, retinoic acid (RA) is added to regulate shared and complementary target genes, enabling the directed conversion of cardiac progenitor cells into atrial myocytes. After observing beating atrial myocytes, they are digested, replated, and maintained in atrial myocyte culture medium. Atrial myocytes with a purity below 90% can be purified.

[0008] Specific steps for implementing the plan:

[0009] iPSC culture: iPSCs should be passaged every 3-5 days and can be passaged when the confluence reaches 70-85%. Before passage, iPSCs should be washed with DPBS, digested with 37°C preheated Tryple for 3 minutes, neutralized with 3-5 volumes of DPBS, and centrifuged at 300g for 5 minutes. iPSCs should be resuspended in StemFlex + 10µM Y-27632 and passaged at a ratio of 1:6.

[0010] Before atrial myocyte differentiation: iPSCs have been passaged 3-5 times and the proliferation rate is relatively stable. Taking a 6-well plate as an example, a 1:6 seeding ratio can reach 70-85% confluence within 48-72 hours, and then iPSCs can be re-plated to start atrial myocyte differentiation.

[0011] Atrial myocyte differentiation steps (using 6-well plate as an example):

[0012] When the confluence of iPSCs reaches 70-85%, they are re-plated at a ratio of 1:3-1:4 after digestion. 48 hours after re-plating, when the confluence of iPSCs reaches more than 85%, differentiation begins and is considered D0.

[0013] The formula of atrial myocyte differentiation basal medium is: RPMI 1640, 2% B27minusinsulin, 80-200uM LAA; the formula of atrial myocyte culture medium is: RPMI 1640, 2% FBS, 2% B27, 1% Pen Strep;

[0014] The formula of atrial myocyte purification medium is: DMEM (no glucose), HEPES, L-Sodium Lactate;

[0015] Step S1, Day 0: Add 3 ml of atrial myocyte differentiation medium to each well, supplemented with 3-6 μM CHIR99021, and continue culturing for 48 hours;

[0016] Step S2, Day 2: Remove the old culture medium and add 3 ml of atrial myocyte differentiation medium to each well, along with 1-3 μM MWnt-C59, and continue culturing for 24 hours.

[0017] Step S3, Day 3: Collect the old culture medium, add 0.5-2 μM RA, mix thoroughly, and add it back to the original culture well. Continue culturing for 24 hours.

[0018] Step S4, Day 4: Remove the old culture medium and add 3 ml of atrial myocyte differentiation medium to each well, supplemented with 0.5-2 μM RA, and continue culturing for 48 hours.

[0019] Step S5, Day 6: Remove the old culture medium and add 3 ml of atrial myocyte differentiation medium to each well. Continue culturing for 48 hours.

[0020] Step S6, Day 8: Remove the old culture medium and add 2 ml of atrial myocyte culture medium to each well. Continue culturing for 48 hours.

[0021] Step S7, Day 10: Remove old culture medium, wash once with DPBS, and add 1 ml of 37°C preheated 0.25% Trypsin-EDTA to each well of a 6-well plate to digest atrial myocytes for 3-6 minutes. Once most cells are observed to detach from the bottom and become rounded, add DMEM F12 containing 10% FBS to terminate digestion. After centrifugation, resuspend in atrial myocyte culture medium plus 10 μM Y-27632 and plate onto a Matrigel-coated 10 cm dish. Change the medium every 48 hours.

[0022] Step S8, Day 12-20: Determine whether purification is needed to improve the purity of atrial myocytes based on the beating behavior of the atrial myocytes. If purification is not required, maintain the culture with atrial myocyte culture medium, changing the medium every 48 hours. If purification is required, remove the old medium on Day 12, add atrial myocyte purification solution, treat for 48 hours, and then switch to atrial myocyte culture medium for long-term maintenance.

[0023] Furthermore, in step S1, after adding a low concentration of 3-6 uM CHIR99021 to the atrial myocyte differentiation medium for 24 hours, less than 50% of the cells will die, which is a normal phenomenon. After 48 hours, the number of cells will increase significantly.

[0024] Furthermore, in step S2, after 1-3 uM Wnt-C59 was added to the atrial myocyte differentiation medium for 24 hours, the cells underwent significant morphological changes and the cell number further increased;

[0025] Furthermore, in step S3, after 1-3uM Wnt-C59 and 0.5-2uM RA were simultaneously added to the atrial myocyte differentiation medium, the cell morphology changed again, and the cells began to show obvious multilayer phenomenon;

[0026] Furthermore, in step S5, spontaneous beating of atrial myocytes can be observed within 48 hours after the atrial myocyte differentiation medium is applied;

[0027] Furthermore, in step S7, according to 6 to 8×10 6 The cells were seeded into 10 cm dishes.

[0028] Furthermore, in step S8, the high-purity atrial myocytes beat in waves, while the non-cardiac myocytes do not beat. The scattered beating atrial myocytes need to be further purified.

[0029] Beneficial effects of the present invention:

[0030] The present invention is a method for atrial myocyte differentiation with high differentiation efficiency and strong universality. It is not only suitable for atrial fibrillation (AF), but also for hereditary arrhythmias such as LQT syndrome, Brugada syndrome, and atrial myocyte differentiation of iPSCs after gene editing to introduce mutations and gene editing to repair mutations. Compared with RPMI 1640+0.5mg / mlhuman albumin+0.2mg / mlLAA as a basal solution for atrial myocyte differentiation, the present invention uses RPMI1640+2%B27minusinsulin+80-200uM LAA as a basal solution for atrial myocyte differentiation. The cell survival rate is significantly improved 48 hours before differentiation, and it is widely applicable to iPSC samples from different sources. In addition, the present invention uses a low concentration of CHIR99021 for 48 hours to reduce the stimulation of cells during the stable transformation of mesoderm cells by iPSCs, thereby improving cell survival rate. The cell state at the initial stage of differentiation is crucial to whether high-quality atrial myocytes can be finally differentiated. During the induction of mesoderm to early cardiac progenitor cells, the present invention utilizes Wnt-C59, rather than the more widely used IWR-1 and IWP2. Wnt-C59 exhibits higher efficiency in inhibiting the Wnt signaling pathway, with an IC50 of only 74 pM, compared to 27 nM for both IWR-1 and IWP2. This indicates that a low concentration of Wnt-C59 can achieve a perfect inhibitory effect. Furthermore, literature reports indicate that cardiomyocytes differentiated with Wnt-C59 exhibit electrophysiological properties closer to mature cardiomyocytes and gene expression more similar to human cardiomyocytes. The present invention utilizes iPSCs to differentiate atrial myocytes, with simple steps and clear functions at each step. It has a wide range of applications, demonstrating high differentiation efficiency when used to differentiate atrial myocytes from iPSCs derived from diverse disease sources. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Flowchart for the differentiation of iPSCs into atrial myocytes.

[0032] Figure 2 Brightfield analysis of atrial myocyte differentiation Day 0-Day 8 using different atrial myocyte differentiation basal solution formulations (using healthy control iPSCs as an example) shows that atrial myocyte conversion efficiency is higher when differentiated using the atrial myocyte differentiation basal solution of the present invention, RPMI 1640 with 2% B27minus insulin and 80-200 μM LAA, and the RPMI 1640 with 0.5 mg / ml human albumin and 0.2 mg / ml LAA commonly used in existing literature, using the same additives.

[0033] Figure 3: Day 0-Day 8 bright field results of atrial myocyte differentiation from healthy control iPSCs, atrial fibrillation patient iPSCs, and gene-edited (mutation introduced) iPSCs showed that high conversion efficiency was demonstrated in the process of atrial myocyte differentiation from iPSCs of three different sources.

[0034] Figure 4 This is a flow cytometry identification diagram of atrial myocytes (taking healthy controls as an example, MLC2a is an atrial myocardial-specific marker). The purity of atrial myocytes is as high as 99%.

[0035] References:

[0036] 1.Davi M.Lyra-Leite,et al.A review of protocols for human iPSCculture,cardiac differentiation,subtype-specification,maturation,and directreprogramming.STAR Protoc.2022,3(3):101560.

[0037] 2.Aleksandra Nijak, et al.Morpho-functional comparison ofdifferentiation protocols to create iPSC-derived cardiomyocytes.Biolopen.2022,11(2):bio059016.

Claims

A method for differentiating atrial myocytes from iPSCs (iPSCs) derived from various sources: RPMI 1640 with 2% B27 minus insulin and 80-200 μM LAA is used as the atrial myocyte differentiation basal medium. Low concentrations of CHIR99021 (3-6 μM) are applied for 48 hours to reduce iPSC cell death and enhance proliferation during the mesoderm-to-mesoderm transition. Wnt-C59 (1-3 μM), a Wnt signaling inhibitor, is then applied to induce the mesodermal differentiation of the mesodermal cells into cardiac progenitor cells. After 24 hours of Wnt-C59 treatment, 0.5-2 μM retinoic acid (RA) is added to regulate shared and complementary target genes. The RA treatment lasts for 72 hours, allowing the cardiac progenitor cells to differentiate into atrial myocytes. After beating is observed, the cells are digested, replated, and maintained in atrial myocyte culture medium. Atrial myocytes with a purity below 90% can be purified. In the atrial myocyte differentiation process of iPSCs as described in claim 1, RPMI 1640 + 2% B27 minus insulin + 80-200uM LAA is used as the atrial myocyte differentiation basal medium to improve cell survival rate and atrial myocyte conversion rate during differentiation. As described in claim 1, at the initiation of differentiation, 3-6 uM CHIR99021 is continuously applied for 48 hours to reduce the number of initial iPSC deaths. The method of claim 1 , wherein 1-3 uM Wnt-C59 is used to induce the transformation of mesodermal cells into cardiomyocytes. As described in claim 1, the mesodermal cells are transformed into a specific myocardial subtype - atrial myocytes through the combined action of 1-3uM Wnt-C59 and 0.5-2uM retinoic acid.

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