A 3d culture method to promote differentiation of stem cells into cardiac lineage cells

Abstract

The application provides a 3D culture method for promoting differentiation of stem cells into heart lineage cells, which adopts four-stage time sequence induction: in stage I, cells are efficiently induced into mesoderm in an insulin-free medium by using BMP4, Activin A and bFGF; in stage II, DKK1 is used to inhibit WNT signal to lock heart lineage, and VEGF165 is added early to start heart endothelium differentiation; in stage III, insulin-containing medium is switched to provide metabolic support, and double-lineage maturation is deepened; in stage IV, bFGF is added to support long-term survival and function maintenance of cells, and the whole process is carried out in 3D embryoid bodies. The application uses inexpensive RPMI1640 basic medium to replace expensive commercial medium, and high-purity heart microtissues containing myocardial and endocardial cells and spontaneously beating can be obtained in about 10 days, which provides an economical and efficient cell source for heart disease research, drug screening and regenerative medicine.

Description

Technical Field

[0001] This invention belongs to the field of cell differentiation technology, and more specifically, relates to a 3D culture method for promoting the differentiation of stem cells into cardiac lineage cells. Background Technology

[0002] Human heart organoids are complex multicellular aggregates, encompassing the transcriptional, functional, and morphological characteristics of cardiac tissue, and play a crucial role in disease mechanism research, drug testing, and regenerative medicine. However, current research on human heart organoids is primarily limited to traditional two-dimensional (2D) cell and animal models, which cannot simulate the 3D microenvironment within the human body, leading to limitations in research results. The rise of three-dimensional (3D) culture technology offers a new approach to addressing this issue. 3D culture systems can better simulate the in vivo microenvironment, enhancing precision medicine by modeling disease and in vitro drug interactions through more accurate simulation of the in vivo microenvironment.

[0003] The emergence of human pluripotent stem cells (hPSCs) has brought unprecedented opportunities to cardiac regenerative medicine. hPSCs can be directed to differentiate into various cardiac lineages, reproducing crucial steps in cardiac development in vitro. However, most current studies on directed differentiation of cardiac lineages focus on monolayer cell adhesion differentiation, resulting in a single cell type, primarily cardiomyocytes, which fails to reflect the three-dimensional complexity of real organs and tissues and neglects the contributions of other non-cardiomyocytes (such as endocardium, epicardium, and cardiac fibroblasts) to cardiac development. While some studies have indicated that induced pluripotent stem cells (iPSCs) can differentiate into cardiac endothelial cells at a two-dimensional level, using inexpensive RPMI 1640 basal cell culture medium, this process cannot simulate the in vivo 3D microenvironment. This lack of 3D environmental support leads to insufficient stem cell differentiation efficiency, functional integrity, or purity of differentiated cells, making it difficult to meet the stringent cell quality requirements for clinical translation. Some studies have also used human embryonic stem cells (hESCs) to differentiate into various cardiac lineage cells via the embryomorphic body pathway. Although this simulates a 3D in vivo environment, the cost of the basal cell culture medium StemPro-34 is relatively high, significantly increasing the cost of differentiating stem cells into cardiac lineage cells. This high cost directly limits the large-scale application of the technology and makes it difficult to meet the practical needs of preclinical research and industrial production.

[0004] The urgent problem to be solved in current human heart organoid culture systems is how to provide a method that can not only improve the differentiation efficiency, functional integrity and purity of stem cells into cardiac lineage cells, but also reduce research costs and meet the needs of clinical applications and research. Summary of the Invention

[0005] To address the problems existing in current technologies, this invention provides a 3D culture method for promoting the differentiation of stem cells into cardiac lineage cells. This method achieves 3D induced differentiation of stem cells into cardiac lineage cells (containing both cardiomyocytes and endothelial cells) in approximately 10 days, providing a new, economical, and efficient pathway for the production of stem cell-derived cardiac lineage cells. This has significant implications for drug screening, cardiac developmental biology, and regenerative medicine research. One objective of this invention is to provide a culture medium system that improves the efficiency of stem cell differentiation into cardiac lineage cells or the functional maturity of cardiac lineage cells. A second objective is to provide a culture method that meets clinical needs while being economical. A third objective is to provide a cardiac lineage structure containing both cardiomyocytes and endothelial cells.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] On one hand, the present invention provides a culture medium composition for promoting the differentiation of stem cells into cardiac lineage cells, comprising the following components according to the stage of use:

[0008] a) Phase I culture medium containing at least one small molecule protein from BMP4, ActivinA, and bFGF;

[0009] b) Phase II culture medium containing at least one small molecule protein from VEGF165 and DKK1;

[0010] c) Phase III culture medium containing at least one small molecule protein from VEGF165 and DKK1;

[0011] d) Phase IV culture medium containing at least one small molecule protein from VEGF165, DKK1, and bFGF.

[0012] Furthermore, the concentration of BMP4 is 50-70 ng / mL, the concentration of Activin A is 8-12 ng / mL, the concentration of bFGF is 4-6 ng / mL, the concentration of VEGF165 is 8-12 ng / mL, and the concentration of DKK1 is 140-160 ng / mL.

[0013] To overcome the contradiction between high cost and low biomimicry in existing technologies, this invention is based on the inexpensive RPMI 1640 culture medium and creatively adopts a four-stage sequential induction culture strategy to achieve the economical, efficient and stable induction of a cardiac lineage structure containing both cardiomyocytes (CM) and cardiac endothelial cells (EC) from human pluripotent stem cells in a three-dimensional (3D) environment. The fundamental logic of this technical solution is derived from the deep simulation of the biological process of embryonic heart development.

[0014] Heart development is not a one-step process, but follows a strict spatiotemporal sequence: pluripotent epidermal cells first differentiate into mesoderm upon receiving specific signals; some of these mesoderm cells, under the influence of local signals, further specialize into cardiac mesoderm / cardiovascular progenitor cells; these progenitor cells then expand and eventually differentiate into various functional terminal cell types (such as cardiomyocytes, endothelial cells, etc.), and form compartmental structures through self-organization. The four-stage culture protocol provided by this invention is a precise mapping and controllable reproduction of this natural process: Stage I uses Phase I culture medium to induce mesoderm; Stage II uses Phase II culture medium to induce mesoderm to specialize into cardiac progenitor cells; Stage III uses Phase III culture medium to initiate dual lineage differentiation; Stage IV uses Phase IV culture medium to achieve terminal maturation and maintenance. Each stage sequentially turns on, off, or adjusts the intensity of key signaling pathways through specific induction culture media, guiding stem cells step by step towards a predetermined endpoint, much like a programmed sequence. This significantly improves the synchronicity, purity, and efficiency of differentiation, and significantly promotes the differentiation of stem cells into high-quality, homogeneous cardiac lineage cells.

[0015] First, this invention utilizes a Phase I culture medium to powerfully initiate the exit of pluripotent stem cells from their pluripotent state, efficiently and synchronously transforming them into mesodermal precursor cells, providing a pure cell source for subsequent heart-specification. Regarding the Phase I culture medium formulation, this invention creatively incorporates a combination of BMP4, Activin A, and bFGF into the basal medium to simulate the signaling environment during embryonic development. BMP4 and Activin A synergistically activate the intracellular SMAD signaling pathway. The synergistic effect of these two signals is the strongest instruction to initiate mesodermal differentiation, directly inhibiting pluripotency genes (such as OCT4) and activating mesodermal marker genes (such as T). bFGF, by activating the MAPK / ERK pathway, provides cells with vigorous proliferation and survival signals, ensuring a sufficient number of cells during differentiation and enhancing cell responsiveness to BMP / Activin signals. The entire induction process takes place in a 3D embryoid body (EB). The cell clusters formed by the EB, with naturally generated cell contact and signal gradients within, more realistically simulate the three-dimensional microenvironment of embryonic development, making mesodermal induction more uniform and efficient, avoiding the asynchronous differentiation problems commonly found in 2D culture.

[0016] Next, mesodermal cells were induced to differentiate into cardiac progenitor cells using Phase II medium, with two inducing factors, DKK1 and VEGF165, introduced into the basal medium. DKK1 blocks WNT signaling by binding to LRP5 / 6 receptors. This inhibition of the WNT pathway at this stage responds to the biological event of WNT signal downregulation promoting heart formation during embryonic development. It locks the developmental potential of mesodermal cells into the cardiovascular lineage, effectively inhibiting their differentiation into other mesodermal pathways such as skeletal muscle and hematopoiesis. Simultaneously, the early introduction of VEGF165 is proactive. By activating the VEGFR2 receptor, it initiates endothelial-specific gene programs (such as CD31 expression), prompting some already oriented cardiac progenitor cells to transition to the epithelial-mesenchymal lineage earlier and shift towards the cardiac endothelial cell lineage. During this stage, insulin-free "RB-" medium is still used to maintain a relatively basal metabolic environment, forcing cells to use energy primarily for fate switching rather than rapid proliferation.

[0017] Then, in stage III, the terminal differentiation program of cardiac and endothelial cells is initiated, providing crucial metabolic transitions for functional maturation. At this point, the basal medium is switched from "RB-" to "RB+", and insulin is added. Insulin, by activating the PI3K-Akt-mTOR pathway, significantly enhances cellular glucose uptake and metabolism, providing sufficient energy for the subsequent synthesis of large amounts of contractile proteins, ion channel proteins, and the initiation of contraction. Simultaneously, the continuous supply of VEGF165 drives further proliferation and migration of cells already biased towards EC differentiation, and they begin to express more mature endothelial markers; while the continuous WNT inhibition by DKK1 helps cardiac progenitor cells maintain the correct differentiation direction and avoid signal disruption.

[0018] Finally, cell culture enters the terminal maturation stage. The core role of the Phase IV medium at this stage is to support the functional maturation of dual-lineage cells and maintain the long-term viability and stability of 3D organoid structures. bFGF is reintroduced into the medium during this stage, which, on the one hand, continuously promotes cell survival and health (especially cardiomyocytes); on the other hand, it may enhance electrical coupling between cardiomyocytes by regulating the expression of connective proteins. The continued action of VEGF165 drives endothelial cells to complete tubular formation and form a preliminary tubular network structure, which is crucial for nutrient transport and metabolic waste removal within the 3D embryoid body and is the foundation for the long-term survival and maturation of tissue-like structures. The retention of DKK1 continues to provide a stable low-WNT environment for the mature phenotype of cardiomyocytes. During this stage, with sufficient energy provided by insulin and the support of bFGF, cardiomyocytes express large amounts of mature structural proteins (such as cTnT) and assemble into sarcomeres with clearly defined light and dark bands, ultimately achieving coordinated and spontaneous rhythmic contractions.

[0019] In summary, this invention designs a logically rigorous and precisely regulated four-stage 3D differentiation scheme by simulating the principles of developmental biology. This scheme, with its unique combination of factors and low-cost basal culture medium, successfully solves the key technical problem of balancing cost, efficiency, and biomimicry in the preparation of cardiac lineage cells. It provides a highly competitive tool and cell source for cardiac regenerative medicine, disease model construction, and drug development, possessing significant scientific value and broad prospects for industrial application.

[0020] Furthermore, the basal medium for the Phase I and Phase II culture media is RB-medium, which is RPMI 1640 basal medium without insulin; the basal medium for the Phase III and Phase IV culture media is RB+medium, which is RPMI 1640 basal medium containing insulin.

[0021] Furthermore, the formulation of the RB- medium includes the following components: RPMI 1640 basal culture medium, insulin-free B27 supplement, L-glutamine, L-ascorbic acid, recombinant human transferrin, thioglycerol, penicillin, and streptomycin; the formulation of the RB+ medium includes the following components: RPMI 1640 basal culture medium, complete B27 supplement, L-glutamine, L-ascorbic acid, recombinant human transferrin, thioglycerol, penicillin, and streptomycin.

[0022] On the other hand, the present invention provides a method for culturing stem cells to promote differentiation into cardiac lineage cells, using a culture medium composition as described above to induce stem cell differentiation.

[0023] Furthermore, the method includes the following steps:

[0024] 1) Culture stem cells to form embryoid bodies;

[0025] 2) The embryoid body was induced to differentiate into the cardiac mesoderm using stage I culture medium;

[0026] 3) Phase II culture medium was used to induce differentiation of cardiac mesodermal embryoids into cardiac progenitor cells;

[0027] 4) Phase III culture medium was used to induce cardiac progenitor cells to differentiate into cardiac lineage cells;

[0028] 5) Phase IV culture medium was used to further induce terminal differentiation of cardiac lineage cells.

[0029] Furthermore, the stem cells are human embryonic stem cells H9; the cardiac lineage cells include at least cardiomyocytes and cardiac endothelial cells.

[0030] In another aspect, the present invention provides cardiac lineage cells cultured by the culture method described above.

[0031] In another aspect, the present invention provides the use of the culture medium composition described above in the preparation of reagents that improve the efficiency of stem cell differentiation into cardiac lineage cells or the functional maturity of cardiac lineage cells.

[0032] Furthermore, this invention provides the application of cardiac lineage cells as described above in drug screening, cardiac developmental biology, and regenerative medicine research.

[0033] The present invention has the following beneficial effects:

[0034] 1. The four-stage sequential induction ensures a high degree of synchronization and targeting of the differentiation process. Phase I establishes a homogeneous mesoderm, Phase II precisely targets cardiac fate and actively induces cardiac endothelial cell differentiation, effectively reducing the occurrence of contaminating cells and non-target differentiation pathways. qPCR data show that the present invention can efficiently produce high-purity, highly active cardiac lineage cells containing both cardiomyocytes and cardiac endothelial cells within 10 days, with a coordinated ratio of the two lineages, meeting the requirements for high-quality cell products.

[0035] 2. The use of inexpensive RPMI 1640 series basal media (RB- / RB+) successfully replaced the expensive commercial media commonly used in cardiac organoid culture (such as StemPro-34, with a cost difference of approximately 100 times). Comparative experiments across all stages confirmed that this invention achieved levels comparable to or even superior to those using expensive media in key aspects such as the expression of cardiac and endothelial cell markers and cell function, truly achieving cost reduction without sacrificing efficiency, thus removing the primary economic obstacle for large-scale production and clinical application.

[0036] 3. 3D embryoid suspension culture not only simulates the three-dimensional space of cell growth and intercellular interactions in vivo, but more importantly, the naturally formed nutrient, oxygen, and growth factor gradients within it are the key physical force driving the spatial self-organization of cardiomyocytes and endothelial cells into tissue-like structures. Cells cultured in this 3D environment exhibit morphology, functional maturity, and gene expression profiles that more closely resemble their real-world in vivo state, far surpassing traditional 2D culture. This provides more reliable predictive data in drug screening, toxicity testing, and disease modeling. Attached Figure Description

[0037] Figure 1 A flowchart illustrating the technical solution provided by this invention.

[0038] Figure 2 Example of human embryonic stem cell H9 morphology in mTeSR1 culture medium.

[0039] Figure 3 The expression levels of cellular markers were detected by qPCR (A) and RT-PCR (B). Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to embodiments. It should be noted that the following embodiments are only used to explain and illustrate this invention and are not intended to limit this invention.

[0041] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0042] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0043] To make this invention easier to understand, some terms used herein are defined and explained below:

[0044] BMP4: Bone morphogenetic protein 4, a member of the TGF-β superfamily.

[0045] Activin A: A member of the TGF-β superfamily.

[0046] bFGF: Basic fibroblast growth factor, also known as FGF2.

[0047] VEGF165: Vascular endothelial growth factor 165, the most important subtype of the VEGF-A family.

[0048] DKK1: Dickkopf-associated protein 1, a classic WNT signaling pathway inhibitor.

[0049] Example 1: The method for promoting stem cell differentiation into cardiac lineage cells provided by the present invention

[0050] This embodiment provides a method for inducing human embryonic stem cells H9 to differentiate into cardiac lineage cells, the procedure is as follows: Figure 1 As shown, the specific steps are as follows:

[0051] (1) Culture of 3D embryoid bodies using human embryonic stem cells H9

[0052] a) Corning diluted 1:200 ® Matrigel ®(Catalog No. 354277) Coat 12-well plates with working solution and incubate at 37°C for at least 30 minutes. Resuscitate H9 cells in Matrigel-coated wells, add mTeSR1 (STEMCELL Technologies, Catalog No. 85850) stem cell culture medium containing 10 µMROCK inhibitor Y-27632. When confluence reaches 70%–80%, discard the mTeSR1 medium, then wash once with calcium- and magnesium-free PBS solution and use ReLeSR... TM The digestive solution digests the cells into small clumps, which are then passaged at a ratio of 1:(5~10). Fresh mTeSR1 medium is replaced daily (preheated to room temperature before each replacement) until the cell confluence reaches about 80%.

[0053] b) H9 cells with 80% confluence were digested into single cells using ACCUTASE (STEMCELL Technologies, catalog number 07920) digestion solution. Cells were counted and seeded at a density of 20,000 cells / well into 96-well U-bottom cell culture plates pre-treated with anti-adhesion solution. Each well was then supplemented with mTeSR1 stem cell culture medium containing 10 µM ROCK inhibitor Y-27632. The plates were centrifuged at 400 rcf for 5 minutes. The plates were then placed in a 37°C CO2 incubator on a shaker and cultured overnight with horizontal shaking at 60 rpm. The next day, uniformly sized and shaped embryoid spheres (…) were observed. Figure 2 ).

[0054] (2) Stage I (D0~D3): Induction of embryoid body to differentiate into cardiac mesoderm

[0055] a) Replace the stem cell culture medium used for culturing embryoids with Phase I culture medium to induce differentiation of embryoids into the cardiac mesoderm stage. The culture time is 3 days. The Phase I culture medium consists of the following: 20 mL RB-induction medium, BMP4, Activin A, and bFGF. The concentration of BMP4 is 60 ng / mL, the concentration of Activin A is 10 ng / mL, and the concentration of bFGF is 5 ng / mL.

[0056] The composition of the RB-induction medium is as follows: RPMI 1640 basal culture medium, 1% insulin-free B27 supplement, 2mM L-glutamine, 50 µg / mL L-ascorbic acid, 150 µg / mL recombinant human transferrin, 50 µg / mL thioglycerol, 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0057] (3) Stage II (D3~D5): Induction of cardiac mesodermal embryoid bodies to differentiate into cardiac progenitor cells

[0058] Replace the Phase I culture medium used in step (2) above for culturing cardiac mesodermal embryoids with Phase II culture medium to induce differentiation of cardiac mesodermal embryoids into cardiac progenitor cells. The culture time is 2 days. The Phase II culture medium consists of 20 mL of RB-induction medium, VEGF165, and DKK1. The concentration of VEGF165 used is 10 ng / mL, and the concentration of DKK1 used is 150 ng / mL. The formulation of RB-induction medium is the same as in step (2).

[0059] (4) Stage III (D5~D7): Inducing cardiac progenitor cells to differentiate into cardiac lineage cells

[0060] Replace the Phase II culture medium used in step (3) above for culturing cardiac progenitor cells with Phase III culture medium to induce cardiac progenitor cells to differentiate into cardiac lineage cells. The culture time is 2 days. The Phase III culture medium consists of 40 mL of RB+ induction medium, VEGF165, and DKK1. The concentration of VEGF165 used is 10 ng / mL, and the concentration of DKK1 used is 150 ng / mL.

[0061] The composition of the RB+ induction medium is as follows: RPMI 1640 basal culture medium, 1% complete B27 supplement, 2 mL glutamine, 50 µg / mL L-ascorbic acid, 150 µg / mL recombinant human transferrin, 50 µg / mL thioglycerol, 100 units / mL penicillin, and 100 μg / mL streptomycin.

[0062] (5) Stage IV (D7 and beyond): Maturation and maintenance of cardiac lineage cells

[0063] Replace the Phase III culture medium used in step (4) above for culturing cardiac progenitor cells with Phase IV culture medium to induce further differentiation of cells into 3D cardiac lineage cells containing both cardiomyocytes and cardiac endothelial cells. The culture time should last for 3 days or more. The Phase IV culture medium consists of the following: 40 mL of RB+ induction medium, VEGF165, DKK1, and bFGF. The concentration of VEGF165 used is 10 ng / mL, the concentration of DKK1 used is 150 ng / mL, and the concentration of bFGF used is 5 ng / mL. The formulation of RB+ induction medium is the same as in step (4).

[0064] The expression levels of relevant markers of differentiated cells in the above steps were detected using qPCR technology, and the results are as follows: Figure 3As shown, the expression level of OCT4, a pluripotency marker of human embryonic stem cells H9, decreased continuously from D0 to D6 and then stabilized. The expression level of T, a marker of mesoderm, increased significantly from D0 to D2 and decreased significantly from D2 to D4, indicating that human embryonic stem cells were successfully induced to differentiate into mesoderm during the early culture stage. The mesoderm was then successfully induced to differentiate into cardiac progenitor cells (the expression level of marker NKX2.5 began to increase from D2). From D3 to D8, the expression levels of cardiomyocyte markers TNNT2 and NKX2.5 and cardiac endothelial cell markers CD31 and CDH5 gradually increased, indicating that human embryonic stem cells H9 were successfully induced to differentiate into cardiac lineage cells containing both cardiomyocytes and cardiac endothelial cells. This demonstrates that the culture method provided by this invention can significantly promote the differentiation of stem cells into cardiac lineage cells.

[0065] Example 2: Screening of culture medium formulations

[0066] To verify the effect of culture medium composition on stem cell differentiation into cardiac lineage cells, this embodiment designed the following comparative experiment for screening basal culture medium and differentiation-inducing factors:

[0067] (1) Composition of culture medium in Phase I

[0068] The culture was carried out according to the experimental procedure of Example 1, except that the culture medium for Phase I was prepared according to the following four formulations:

[0069] Formula 1: RB-induction medium, 60 ng / mL BMP4, 10 ng / mL Activin A, 5 ng / mL bFGF

[0070] Formula 2: RB-induction medium, 60 ng / mL BMP4, 3 μM CHIR99021, 5 ng / mL bFGF

[0071] Formula 3: IMDM basal medium, 60 ng / mL BMP4, 10 ng / mL Activin A, 5 ng / mL bFGF

[0072] The expression levels of cardiomyocyte markers TNNT2 and NKX2.5 and endothelial cell markers CD31 and CDH5 induced by the above three schemes were detected by qPCR. The results are shown in Table 1.

[0073] Table 1. Effects of Phase I culture medium composition on the differentiation degree of cardiomyocytes and cardiac endothelial cells.

[0074]

[0075] Note: Data represent relative expression folds with respect to undifferentiated hPSCs after calibration, consistent with expression levels in the early differentiation stage, and are expressed as mean ± standard deviation.

[0076] As shown in Table 1, the expression of markers in cardiomyocytes and endothelial cells induced by formulation 1 of the present invention was significantly higher than that in other control groups, demonstrating its superiority as the optimal initiation scheme. Compared with formulation 2 using the classic WNT activator CHIR99021, the combination of BMP4 and Activin A in formulation 1 showed a better synergistic effect. Although CHIR99021 can strongly activate the WNT pathway to drive mesoderm formation, the experimental results of this embodiment show that the expression fold of markers in terminal cardiomyocytes and endothelial cells in formulation 1 was higher than that in formulation 2, and the development of the two lineages was more balanced. This indicates that in the 3D embryoid (EB) culture system of the present invention, BMP4 and Activin A may induce a more homogeneous mesodermal cell population that is more suitable for development into the cardiovascular lineage by synergistically activating the SMAD signaling pathway.

[0077] Secondly, the RB-induced differentiation medium provided by this invention is a key component for the successful initiation of the differentiation process. Formula 3, using only IMDM basal medium, even with the addition of the same growth factors, only showed slightly higher than background levels in its terminal cardiac marker expression, a significant difference compared to Formula 1. This indicates that the RB-induced differentiation medium designed in this invention is not simply a nutrient carrier; its specific components and physicochemical properties create the optimal initial microenvironment for efficient signal transduction of BMP4 and ActivinA, an indispensable condition for efficient mesodermal induction. In summary, the Phase I medium provided by this invention, through a proprietary basal medium combined with specific factors, achieves efficient, uniform, and well-directed mesodermal induction, laying an irreplaceable and solid foundation for successfully obtaining high-purity cardiac dual-lineage cells in subsequent stages.

[0078] (2) Composition of Phase II culture medium

[0079] The culture was carried out according to the experimental procedure of Example 1, except that the culture medium for Phase II was prepared according to the following four formulations:

[0080] Formula 4: RB-induction medium, 10 ng / mL VEGF165, 150 ng / mL DKK1;

[0081] Formula 5: RB-induction medium, 10 ng / mL VEGF165;

[0082] Formula 6: RB-induction medium, 150 ng / mL DKK1;

[0083] Formula 7: RB-induction medium, 10 ng / mL VEGF165, 5 μM conventional WNT inhibitor IWP-2;

[0084] The expression levels of cardiomyocyte markers TNNT2 and NKX2.5 and endothelial cell markers CD31 and CDH5 induced by the above four methods were detected by qPCR. The results are shown in Table 2.

[0085] Table 2. Effects of Phase II culture medium composition on the differentiation degree of cardiomyocytes and cardiac endothelial cells.

[0086]

[0087] Note: Data represent relative fold increases with respect to the internal reference gene GAPDH after calibration, expressed as mean ± standard deviation.

[0088] qPCR data showed that the expression levels of cardiomyocyte markers TNNT2 and NKX2.5 and endothelial cell markers CD31 and CDH5 induced by Formula 4 were significantly higher than those in other control groups, demonstrating that it can drive simultaneous and efficient differentiation of both lineages. The single-component control groups (Formulas 5 and 6) confirmed the indispensability of the two components: when only VEGF165 was present (Formula 5), ​​endothelial differentiation was still acceptable, but myocardial differentiation was severely impaired; when only DKK1 was present (Formula 6), myocardial differentiation was still acceptable, but endothelial differentiation was almost inhibited. This indicates that VEGF165 is responsible for actively driving stem cell differentiation into endothelial cells, while DKK1 locks the cell population into the cardiac lineage by inhibiting WNT signaling. The two are complementary in function and neither can be omitted.

[0089] Furthermore, formulation 7, which uses the conventional WNT inhibitor IWP-2 to replace DKK1, showed better dual-lineage differentiation than the single-component group, but significantly lower than formulation 4 of this invention. This result proves that the role of DKK1 cannot be simply and equivalently replaced by any WNT inhibitor. In the 3D culture system of this invention and in synergy with VEGF165, DKK1 exhibits better compatibility and efficacy.

[0090] (3) Composition of Phase III culture medium

[0091] The culture was carried out according to the experimental procedure of Example 1, except that the Phase III culture medium was prepared according to the following 5 formulations:

[0092] Formula 8: RB+ induction medium, 10 ng / mL VEGF165, 150 ng / mL DKK1;

[0093] Formula 9: RB+ induction medium, 10 ng / mL VEGF165;

[0094] Formula 10: RB+ induction medium, 150 ng / mL DKK1;

[0095] Formula 11: RB+ induction medium, 10 ng / mL VEGF165, 5 μM conventional WNT inhibitor IWP-2;

[0096] Formula 12: RB-induction medium, 10 ng / mL VEGF165, 150 ng / mL DKK1

[0097] The expression levels of cardiomyocyte markers TNNT2 and NKX2.5 and endothelial cell markers CD31 and CDH5 induced by the above five schemes were detected by qPCR. The results are shown in Table 3.

[0098] Table 3. Effects of Phase III culture medium composition on the differentiation degree of cardiomyocytes and cardiac endothelial cells.

[0099]

[0100] Note: Data represent relative fold increases with respect to the internal reference gene GAPDH after calibration, expressed as mean ± standard deviation.

[0101] As shown in Table 3, among the cardiomyocytes and endothelial cells induced by the five formulations, formulation 8 showed the most prominent expression levels of TNNT2, NKX2.5, CD31, and CDH5. Compared to formulation 8, the lack of VEGF165 (formulation 10) led to a sharp decrease in the expression levels of CD31 and CDH5, demonstrating the absolute necessity of VEGF165 for terminal maturation of endothelial cells. The lack of DKK1 (formulation 9) significantly decreased the expression levels of the cardiac markers TNNT2 and NKX2.5, indicating that DKK1 continuously inhibits WNT signaling in stage III and is equally indispensable for the stable maturation of cardiomyocytes. Furthermore, comparing the effects of formulations 8 and 12 shows that replacing the RB-induction medium with the RB+ induction medium in stage III significantly improves the functional maturation of cardiomyocytes and endothelial cells. This result proves that adding insulin to provide metabolic support in stage III is crucial.

[0102] Furthermore, a comparison of the results of formulations 8 and 11 reveals that adding DKK1 to the phase III culture medium has significant advantages over using the conventional inhibitor IWP-2, further demonstrating that the effect of DKK1 in the 3D culture system of this invention is specific and its mechanism of action is more suitable for promoting the synergistic maturation of the two lineages.

[0103] (4) Composition of culture medium in phase IV

[0104] The culture was carried out according to the experimental procedure of Example 1, except that the culture medium for phase IV was prepared according to the following four formulations:

[0105] Formula 13: RB+ induction medium, 10 ng / mL VEGF165, 150 ng / mL DKK1, 5 ng / mL bFGF;

[0106] Formula 14: RB+ induction medium, 10 ng / mL VEGF165, 150 ng / mL DKK1

[0107] Formula 15: RB+ induction medium, 150 ng / mL DKK1, 5 ng / mL bFGF

[0108] Formula 16: RB+ induction medium, 10 ng / mL VEGF165, 5 ng / mL bFGF

[0109] The expression levels of cardiomyocyte markers TNNT2 and NKX2.5 and endothelial cell markers CD31 and CDH5 induced by the above four methods were detected by qPCR. The results are shown in Table 4.

[0110] Table 4. Effects of Phase IV culture medium composition on the differentiation degree of cardiomyocytes and cardiac endothelial cells.

[0111]

[0112] Note: Data represent relative fold increases with respect to the internal reference gene GAPDH after calibration, expressed as mean ± standard deviation.

[0113] Table 4 shows that the expression levels of all markers in Formula 13 were the highest and significantly better than those in the control groups, confirming that Formula 13 is the optimal combination for supporting the long-term functional maturation of cardiac microtissues. Comparing Formula 13 and Formula 14, the lack of bFGF led to a significant decrease in the expression levels of all four markers. This is because bFGF exerts a powerful function of promoting survival, inhibiting apoptosis, and supporting cell metabolism by activating pathways such as MAPK / ERK. In stage IV, bFGF ensures that cells that have undergone rapid differentiation can maintain vigorous vitality, thereby completing the final maturation process. Furthermore, comparing Formulas 13, 15, and 16 also revealed that the lack of VEGF165 or DKK1 significantly reduced the expression levels of endothelial cell markers and cardiomyocyte markers. This result indicates that even at maturity, VEGF165 remains crucial for maintaining the endothelial cell phenotype, preventing its degeneration, and promoting vascular networking; simultaneously, continuous WNT inhibition by DKK1 is still necessary to maintain the mature phenotype and electrophysiological stability of cardiomyocytes. Once removed, cardiomyocytes may undergo dedifferentiation or functional disorders.

[0114] The above results indicate that VEGF165, DKK1, and bFGF have a significant synergistic effect on the maturation stage of cardiac lineage cells. The three constitute a stable and synergistic support system, and the absence of any component will lead to a decrease in maturity or lineage imbalance. None of them can be omitted.

[0115] Example 3: Comparison of the 3D culture method provided by the present invention with traditional methods

[0116] To verify the significant advantages of the 3D culture method for cardiac lineage cells provided by this invention, this embodiment compares the culture method of Example 1 with the traditional 3D culture method. The specific groupings are as follows:

[0117] Experimental group: Human embryonic stem cells H9 were induced to differentiate into cardiac lineage cells according to the culture method of Example 1;

[0118] Control Group 1: Human embryonic stem cells H9 were induced to differentiate into cardiac lineage cells according to the method described in the existing technology "Human cardiovascular progenitor cells develop from a KDR+ embryonic-stem-cell-derived population (doi:10.1038 / nature06894)". The culture medium formula was as follows: StemPro-34 basal medium was used throughout the induction process. In the first stage, 10 ng / mL BMP4, 3 ng / mL ActivinA, and 5 ng / mL bFGF were added to induce differentiation of mesoderm cells. In the second stage, 150 ng / mL DKK1 and 10 ng / mL VEGF were added to induce differentiation of cardiac group cells. In the third stage, 150 ng / mL DKK1, 10 ng / mL VEGF, and 5 ng / mL bFGF were added to induce differentiation of terminally differentiated cardiac lineage cells.

[0119] Control Group 2: Following the method described in the existing technology "BMP10 Signaling Promotes the Development of Endocardial Cells from Human Pluripotent Stem Cell-Derived Cardiovascular Progenitors (doi:10.1016 / j.stem.2020.10.003.)," human embryonic stem cells H9 were induced to differentiate into cardiac lineage cells. The culture medium formulation was as follows: StemPro-34 basal medium was used throughout the induction process. In the first stage, 10 ng / mL BMP4, 6 ng / mL ActivinA, and 5 ng / mL bFGF were added to induce differentiation in the mesoderm; in the second stage, 50 ng / mL bFGF was added to induce differentiation in the cardiac group cells; and in the third stage, 50 ng / mL bFGF and 10 ng / mL BMP10 were added to induce differentiation in the terminally differentiated cardiac lineage cells.

[0120] After induction, the expression levels of cardiomyocyte markers TNNT2 and NKX2.5 and endothelial cell markers CD31 and CDH5 in each group were detected by qPCR. The results are shown in Table 5.

[0121] Table 5 Comparison between the present invention and traditional methods

[0122]

[0123] The comparative results show that the expression of biomarkers in cardiomyocytes and endothelial cells differentiated using the method provided by this invention is at an optimal level. Although the traditional method uses expensive StemPro-34 medium, the induction effect of control group 1 is slightly lower than that of this invention. While control group 2 achieves endothelial cell differentiation, cardiomyocyte differentiation is severely insufficient. This directly proves that the medium combination provided by this invention is significantly superior to the traditional 3D method in driving dual-lineage cell differentiation. Meanwhile, the cost of commercially available mediums such as StemPro-34 is typically tens to hundreds of times higher than RPMI 1640. The method of this invention achieves excellent results while reducing the cost of medium to an extremely low level, demonstrating significant advantages in terms of economy and efficiency.

[0124] Example 4: Culture of human induced pluripotent stem cells (iPSCs)

[0125] To verify that the culture medium and culture method provided by this invention are universal and can also be used to induce differentiation of other cell lines into cardiac lineage cells, this embodiment further uses human induced pluripotent stem cells (iPSCs) for verification.

[0126] The method for promoting stem cell differentiation into cardiac lineage cells provided in this embodiment is basically the same as that in Example 1, except that (1) human embryonic stem cells H9 are replaced with human induced pluripotent stem cells iPSCs; (2) the concentrations of small molecules used in the culture media for phases I, II, III and IV are as follows:

[0127] a) Phase I culture medium: 50 ng / mL BMP4, 12 ng / mL Activin A, 6 ng / mL bFGF;

[0128] b) Phase II culture medium: 8 ng / mL VEGF165, 145 ng / mL DKK1;

[0129] c) Phase III culture medium: 8 ng / mL VEGF165, 145 ng / mL DKK1;

[0130] d) Phase IV culture medium: 8 ng / mL VEGF165, 145 ng / mL DKK1, 6 ng / mL bFGF.

[0131] The expression levels of biomarkers T, TNNT2, NKX2.5, CD31, and CDH5 were detected using qPCR technology, and the results are shown in Table 6.

[0132] Table 6. Expression levels of cellular markers for human induced pluripotent stem cell (iPSC) differentiation.

[0133]

[0134] Note: Data represent relative fold increases with respect to the internal reference gene GAPDH after calibration, expressed as mean ± standard deviation.

[0135] As shown in Table 6, with the extension of culture induction time, the expression level of cardiac mesodermal marker T showed a trend of first increasing and then decreasing. The expression levels of cardiomyocyte markers TNNT2 and NKX2.5, and cardiac endothelial cell markers CD31 and CDH5 gradually increased from D0 to D8, with smaller fluctuations after D8. This indicates that with the change in culture stage, human induced pluripotent stem cells (iPSCs) were first successfully induced to differentiate into cardiac mesoderm. Subsequently, the cardiac mesoderm successfully differentiated into cardiomyocytes and cardiac endothelial cells under the induction of phase II and phase III culture media. The functions of these two cell types were maintained in phase IV culture media. This result proves that the culture medium and method provided by this invention can not only be used for the induction and differentiation of human embryonic stem cells H9, but also effectively promote the differentiation of human induced pluripotent stem cells (iPSCs) into cardiac lineage cells, possessing broad applicability.

[0136] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any modifications and alterations made by those skilled in the art without departing from the spirit and scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for promoting the differentiation of stem cells into cardiac lineage cells using 3D suspension culture, characterized in that, It consists of the following steps: 1) Culture stem cells to form embryoid bodies; 2) The embryoid body was induced to differentiate into the cardiac mesoderm using stage I culture medium; 3) Phase II culture medium was used to induce differentiation of cardiac mesodermal embryoids into cardiac progenitor cells; 4) Phase III culture medium was used to induce cardiac progenitor cells to differentiate into cardiac lineage cells; 5) Phase IV culture medium was used to further induce terminal differentiation of cardiac lineage cells; The Phase I culture medium consists of RB-medium, 50-70 ng / mL BMP4, 8-12 ng / mL Activin A, and 4-6 ng / mL bFGF; The Phase II culture medium consists of RB-medium, 8-12 ng / mL VEGF165, and 140-160 ng / mL DKK1; The Phase III culture medium consists of RB+ medium, 8-12 ng / mL VEGF165, and 140-160 ng / mL DKK1; The Phase IV culture medium consists of RB+ medium, 8-12 ng / mL VEGF165, 140-160 ng / mL DKK1, and 4-6 ng / mL bFGF; The RB-medium consists of the following components: RPMI 1640 basal culture medium, insulin-free B27 supplement, L-glutamine, L-ascorbic acid, recombinant human transferrin, thioglycerol, penicillin, and streptomycin. The RB+ medium is formulated with the following components: RPMI 1640 basal medium, complete B27 supplement, L-glutamine, L-ascorbic acid, recombinant human transferrin, thioglycerol, penicillin, and streptomycin. The stem cells mentioned are human pluripotent stem cells; The cardiac lineage cells include cardiomyocytes and cardiac endothelial cells; The culture conditions for the culture method are as follows: cultured in a CO2 incubator at 37°C.

2. The 3D suspension culture method as described in claim 1, characterized in that, The human pluripotent stem cells mentioned are human embryonic stem cells H9 or human induced pluripotent stem cells.

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