Methods and applications for improving iPSC differentiation efficiency

Abstract

The present invention provides a method and application for improving the differentiation efficiency of iPSCs. This method combines screening-based subculture with Rock inhibitor-retaining differentiation initiation to achieve a comprehensive improvement in the induction efficiency of different 2D / 3D derivatives of endoderm, ectoderm, and mesoderm origin.

Description

Technical Field

[0001] The present invention belongs to the field of cell culture technology, and in particular relates to a method for improving iPSC differentiation efficiency and its application. Background Art

[0002] Since 2006, when S. Yamanaka's team in Japan first reprogrammed somatic cells into induced pluripotent stem cells (iPSCs) by introducing four retrotransposition factors, researchers worldwide have, over the past 20 years, established methods for differentiating iPSCs into various 2D and 3D human derivatives based on in vitro induction of developmental signals. These include 2D cell types such as hepatocytes, cardiomyocytes, and pancreatic islets, as well as 3D organoids such as the liver, lung, and kidney. Compared to traditional cell / tissue culture, this novel technique overcomes the limitation of source resources while reproducing key functional properties of the corresponding in vivo components in vitro. Consequently, this technique has broad applications in disease modeling, drug screening, cell therapy, and organ repair.

[0003] With the advancement of industrialization, the low yield and high production cost caused by insufficient induction efficiency have become the main shackles restricting large-scale transformation and need to be overcome urgently. At present, the means to improve induction efficiency mainly focus on adjusting technical parameters during the differentiation process. For example, in 2D hepatocyte differentiation, Y Nagamoto et al. increased the induction efficiency by overexpressing the hepatocyte nuclear factor HNF1a and achieved ALB + Improvement of liver cell purity. For example, in the induced differentiation of 3D lung organoids, HW Snoeck's team enhanced the efficiency of anterior lateralization induction by adding a Wnt signal inhibition step during the anterior lateralization induction stage, which enabled lung progenitor cells (NKX2.1 + ) yield was significantly improved, thereby increasing the yield of lung organoids.

[0004] However, as the starting point of induced differentiation, although the quality of iPSCs directly determines the differentiation efficiency of derivatives, source optimization such as pre-differentiation acclimation culture and differentiation inoculation is still a neglected link; there are few reports on improving induction efficiency based on improvements in iPSC culture technology. Summary of the Invention

[0005] In view of this, the present invention aims to propose a method for improving iPSC differentiation efficiency and its application. This method is a method for optimizing conditions before differentiation, combining screening-based subculture and Rock inhibitor-retaining differentiation initiation to achieve a universal improvement in the induction efficiency of different 2D / 3D derivatives such as endoderm-derived, ectoderm-derived, and mesoderm-derived.

[0006] To achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] A method for improving iPSC differentiation efficiency, comprising the following steps:

[0008] (1) Screening subculture:

[0009] iPSCs that have been routinely passaged are briefly digested with digestive enzymes for 1.5-2 minutes, undifferentiated cells are selectively recovered, and then passaged continuously for at least five generations, with brief digestion and undifferentiated cell recovery performed at each generation. This gradually eliminates cells that are relatively insensitive to digestive enzymes and enriches for enzyme-sensitive iPSCs, which have higher differentiation efficiency.

[0010] (2) Rock inhibitor retention differentiation initiation:

[0011] The iPSCs collected at the end of step (1) are inoculated into a culture medium containing Rock inhibitor to proliferate and differentiate until differentiation is initiated.

[0012] In some embodiments, in step (1), each time the cells are subcultured until the confluence reaches 70-80%, a digestive enzyme is used for a short digestion.

[0013] The digestive enzymes were preheated to 37°C before use.

[0014] In some embodiments, in step (2), the collected iPSCs are first restored to conventional digestion conditions and the cells are collected, and then inoculated and proliferated, with an inoculation density of 1-3×10 5 pieces / cm 2 .

[0015] Conventional digestion conditions are: digestion with digestive enzymes for 5-10 minutes until all cells are dissociated.

[0016] In some embodiments, in step (2), iPSCs are proliferated in a culture medium containing a Rock inhibitor for 24-72 hours, with the culture medium being replaced every 24 hours until the starting confluence required by the corresponding differentiation protocol is met.

[0017] In some embodiments, before performing step (1), bright field morphology and pluripotency marker quality testing are required to select iPSCs of qualified quality for the experiment.

[0018] Furthermore, the quality inspection standards are:

[0019] Ⅰ. Microscopic examination using an optical microscope: if >90% of the cells exhibit rounded, tightly connected, typical iPSC morphology, and <10% of the cells show signs of differentiation, the cells are considered to have passed morphological examination.

[0020] II. Perform immunofluorescence staining of candidate iPSCs using antibodies against the pluripotency markers OCT4 and NANOG. If the double-positive rate is ≥85%, the candidate iPSCs are considered to have passed the pluripotency test.

[0021] If all the above are inspected, the quality is considered qualified.

[0022] In some embodiments, the Rock inhibitor includes Y-27632 and Thiazovivin, and the concentration of the Rock inhibitor is 2-50 μM.

[0023] Among them, Rock inhibitors include but are not limited to Y-27632 and Thiazovivin, and may also be other Rock inhibitors that can achieve the purpose of the present invention, which are only a limited list; preferably, the Rock inhibitor may be Y-27632.

[0024] Preferably, the concentration of Y-27632 is 10-50 μM, non-limiting examples include 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, more preferably 10 μM;

[0025] The concentration of thiazovivin is 2-10 μM, and non-limiting examples include 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, and 10 μM.

[0026] The present invention also provides an application of the above-mentioned method for improving iPSC differentiation efficiency, which is applied to improving the differentiation efficiency of 2D derivative differentiation and 3D derivative differentiation.

[0027] Furthermore, 2D derivative differentiation includes endoderm 2D derivative differentiation, mesoderm 2D derivative differentiation and ectoderm 2D derivative differentiation, and the average differentiation efficiency of 2D derivative differentiation is increased by more than 10%.

[0028] Furthermore, 3D derivative differentiation includes endoderm 3D derivative differentiation, mesoderm 3D derivative differentiation and ectoderm 3D derivative differentiation, and the average differentiation efficiency of 3D derivative differentiation is increased by more than 10%.

[0029] Compared with the existing technology, the iPSC differentiation efficiency improvement method and application of the present invention have the following advantages:

[0030] (1) The method for improving the iPSC differentiation efficiency described in the present invention combines screening-based subculture and Rock inhibitor-retention-based differentiation initiation, significantly improving the differentiation efficiency. For example, the average induction efficiency of 2D derivatives increased by 12.4%, and the average efficiency of 3D derivatives increased by 14.4%, with the differentiation efficiency both indicating greater than 10%. The method has universal applicability to iPSC derivatives, that is, it is applicable to the induction and differentiation of 2D and 3D derivatives of iPSC endodermal, mesoderm, and ectoderm sources.

[0031] (2) In the screening-type passage culture, the present invention gradually screens out cells that are relatively insensitive to digestive enzymes through continuous short-term passage, thereby achieving the enrichment of enzyme-sensitive iPSCs. Studies have shown that these enzyme-sensitive iPSCs have higher differentiation efficiency.

[0032] (3) In the present invention, Rock inhibitor retention differentiation initiation was originally used for iPSC seeding to increase the adhesion rate and thus improve cell viability, and was removed the day after seeding. However, in the present invention, it was found that the presence of Rock inhibitor can maintain a small distance between iPSC cells, changing the previous classic clonal tight connection state to a small compartment state, thereby allowing the cells to respond synchronously to the induction factor, thereby improving the differentiation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings, which constitute part of the present invention, are provided to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are provided to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0034] Figure 1 Flowchart of methods for improving iPSC differentiation efficiency;

[0035] Figure 2 Immunofluorescence staining for iPSC morphology and pluripotency markers, scale bar = 200 μm;

[0036] Figure 3 Bright field morphology of iPSCs after 24 hours of endoderm differentiation after a single classical passage and screening passage; scale bar = 200 μm;

[0037] Figure 4 Quantitative analysis and comparison of cell confluence after passage 1 using the two methods; n = 4 biological replicates; calculated using Image J software: cell confluence (%) = (full-frame area - exposed well plate area) / full-frame area × 100%, n = 3 biological replicates; **, P < 0.01;

[0038] Figure 5Bright field morphology and quantitative results of cell confluence after 24 hours of endoderm differentiation of iPSCs after continuous classical and screening passages; scale bar = 200 μm;

[0039] Figure 6 Comparison of the quantitative analysis of cell confluence 24 h after endoderm differentiation of iPSCs after continuous classical passage and selection passage; n = 4 biological replicates; **, P < 0.01; ***, P < 0.001;

[0040] Figure 7 After continuous screening and passage, iPSCs were differentiated into endoderm for 72 hours and SOX17 was detected by cytometry. + Cell ratio, n = 3 biological replicates;

[0041] Figure 8 After 5 classic passages and screening passages, iPSCs were differentiated into endoderm for 72 hours and SOX17 was detected by cytometry. + Cell ratio; n = 10 biological replicates; ***, P < 0.001; SOX17, endoderm marker;

[0042] Figure 9 Comparison of flow cytometry results of three differentiation methods; n = 10 biological replicates; **, P < 0.01; ***, P < 0.001;

[0043] Figure 10 Comparison of the differentiation efficiency of iPSCs into 2D hepatocytes, ALB, hepatocyte marker; n = 10 biological replicates; ***, P < 0.001;

[0044] Figure 11 Comparison of the differentiation efficiency of iPSCs into 2D macrophages, CD14, a macrophage-specific marker; n = 10 biological replicates; ***, P < 0.001;

[0045] Figure 12 Comparison of the differentiation efficiency of iPSCs into 2D GABAergic neurons; GABA, a GABAergic neuron-specific marker; n = 10 biological replicates; **, P < 0.01;

[0046] Figure 13 Comparison of the differentiation efficiency of iPSCs into 3D intestinal organoids; CDX2, a broad-spectrum marker of intestinal epithelium; SATB2, a specific marker of colonic epithelium; n = 10 biological replicates; **, P < 0.001;

[0047] Figure 14Comparison of the differentiation efficiency of iPSCs into 3D cardiac organoids; cTNT, a cardiomyocyte-specific marker; n = 10 biological replicates; ***, P < 0.001;

[0048] Figure 15 Comparison of the differentiation efficiency of iPSCs into 3D brain organoids. SOX2 is a neuroepithelial marker. n = 10 biological replicates. **, P < 0.01. DETAILED DESCRIPTION

[0049] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0050] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0051] This method is applicable to the classic iPSC culture system based on mTeSR1 / Matrigel. Some of the reagents used in the present invention are shown in Table 1.

[0052] Example 1 iPSC differentiation

[0053] like Figure 1 As shown, the method for improving iPSC differentiation efficiency includes the following steps:

[0054] 1. Select iPSCs of qualified quality:

[0055] Candidate iPSCs were selected and quality checked by bright field morphology and pluripotency markers:

[0056] 1) Microscopic examination using a light microscope: cells are considered to have passed morphological inspection if >90% of the cells exhibit round, tightly connected, and typical iPSC morphology, and <10% of the cells show signs of differentiation.

[0057] 2) Perform immunofluorescence staining of candidate iPSCs using antibodies against the pluripotency markers OCT4 and NANOG. If the double-positive rate is ≥85%, the candidate iPSCs are considered to have passed the pluripotency test.

[0058] If all of the above pass the inspection, it is considered to be of qualified quality and can be selected for subsequent operations.

[0059] The test results are as follows Figure 2As shown, the upper picture shows the iPSC morphology. It can be seen that there are no recognizable signs of differentiation, and the cells are round and tightly connected, which are typical morphological characteristics of iPSCs; the four pictures at the bottom are immunofluorescence staining assays of pluripotency markers, and the double-positivity rate of the pluripotency markers OCT4 and NANOG is 86%.

[0060] Therefore, the quality was judged to be qualified, and the cells were selected and screened for passage.

[0061] 2. Screening subculture:

[0062] Take qualified iPSCs and continuously screen sensitive iPSCs through short-term digestion method:

[0063] 1) Short-term digestion:

[0064] When the cells reach the standard confluence (70-80%) for passage, digest them using Accutase preheated at 37°C for 2 minutes. Only iPSCs that have not differentiated, are completely dissociated, and are considered more sensitive to digestive enzymes during this period are collected and expanded. Any remaining undissociated cells are considered relatively insensitive or less sensitive and are discarded.

[0065] 2) Continuous screening:

[0066] After the collected cells reach 70-80% confluence, repeat step 1) until 5 consecutive passages have been performed. This completes the screening of sensitive iPSCs and allows for differentiation seeding.

[0067] 3. Rock inhibitor-retaining differentiation initiation

[0068] Take sensitive iPSCs and inoculate them into differentiation plates. Keep Rock inhibitor during the proliferation process until differentiation is initiated:

[0069] 1) iPSC collection:

[0070] At the corresponding differentiation passage, resume standard digestion conditions, i.e., digest with Accutase for 5-10 minutes to completely dissociate and collect all cells. The specific digestion time varies depending on the iPSC cell line.

[0071] 2) Inoculation of differentiation plates:

[0072] After resuspending in mTeSR1 culture medium containing 10 μM Rock inhibitor, 1×10 5 pieces / cm 2 Specifically, the Rock inhibitor may be Y-27632.

[0073] 3) Initiate differentiation:

[0074] Wait for proliferation for 24-72 hours, and replace the culture medium containing 10 μM Rock inhibitor every 24 hours until the starting confluence required by the corresponding differentiation protocol is met.

[0075] The proliferation time is based on the target starting confluence required by different differentiation protocols, and different proliferation times are required.

[0076] Verification 1: Effect of screening passages

[0077] To explore the role of screening passages, a parallel group was established, initially using iPSCs to differentiate into endoderm cells (D'Amour KA, Agulnick AD, Eliazer S, et al. Efficient differentiation of human embryonic stem cells to definitive endoderm[J]. Nature biotechnology, 2005, 23(12): 1534-1541.) Following this differentiation method, iPSCs from both passages were grown to 90% confluence to initiate differentiation, and the differentiation process was analyzed.

[0078] Classical passaging: Digest with Accutase for 5-10 minutes, collect all cells and continue culturing.

[0079] Screening and passage: The digestion step was performed for only 2 minutes, and the corresponding dissociated cells were collected and continued to be cultured.

[0080] The analysis results are as follows Figure 3-Figure 8 As shown:

[0081] like Figure 3-Figure 4 This analysis was performed after a single classical passage and screening passage. Compared to classical passage, iPSCs obtained by short-time digestion in the screening passage had lower cell confluence after 24 hours (average 83.3% vs 70.9%). This result suggests that iPSCs obtained through short-time digestion selection, which are more sensitive to Accutase, are also more sensitive to the cytokine Activin A.

[0082] like Figure 5-Figure 8 Analysis after continuous classical passage and screening passage. Unlike classical passage, continuous screening passage gradually increases the differentiation sensitivity and remains stable after 5 passages ( Figure 5-Figure 6 Flow cytometry analysis after 72 hours of differentiation showed that the increased sensitivity caused by continuous screening and passage can be converted into an increase in the efficiency of endoderm differentiation, and the two are generally positively correlated ( Figure 6-Figure 7Finally, the comparison results based on 10 induced differentiation experiments showed that after 5 passages, the screening passage increased the differentiation efficiency of endoderm cells by about 7.2% ( Figure 8 ).

[0083] Verification of the effect of 2 Rock inhibitors

[0084] Using the aforementioned endoderm differentiation method, the retention effect of Rock inhibitor was investigated. Three parallel groups were set up: iPSCs derived from classical passaging were differentiated with Rock inhibitor withdrawal, iPSCs derived from classical passaging were differentiated with Rock inhibitor retention, and iPSCs derived from screening passaging were differentiated with Rock inhibitor retention.

[0085] Classic passaging method: Digest with Accutase for 5-10 minutes, collect all cells and continue to culture; after 5 consecutive passaging, iPSCs are obtained.

[0086] Screening and passage method: digest for 2 minutes, collect only the corresponding detached cells and continue to culture; after 5 consecutive passages, iPSCs are obtained.

[0087] Withdraw Rock-i (Rock inhibitor): 24 hours after inoculation of the differentiation plate, remove the Rock inhibitor and continue culturing until the cells reach 90% starting confluence.

[0088] Rock-i (Rock Inhibitor) Retention: Retain Rock Inhibitor after inoculating the differentiation plate and continue culturing to 90% starting confluence.

[0089] The Rock-i inhibitor of choice was 10 μM Y-27632.

[0090] Flow cytometry results after differentiation Figure 9 As shown in the figure, using classical iPSC passage, Rock inhibitor retention increased endoderm differentiation efficiency by 5.2%, while combined with selection passage, it increased differentiation efficiency by 11.9%. This demonstrates that, in addition to selection passage, Rock inhibitor retention further enhances the efficiency of endoderm differentiation of iPSCs.

[0091] The following will use different applications to illustrate the broad effect of this method on improving the differentiation efficiency of iPSC-derived 2D and 3D derivatives.

[0092] Application 1 Endoderm 2D derivatives

[0093] To verify the effectiveness of this method in enhancing the differentiation efficiency of 2D endoderm derivatives, we conducted a validation study based on a protocol for directed differentiation of iPSCs into 2D hepatocytes (Sullivan GJ, Hay DC, Park IH, et al. Generation of functional human hepatic endoderm from human induced pluripotent stem cells[J]. Hepatology, 2010, 51(1): 329-335.). Based on the requirements of this method, differentiation was initiated after iPSCs proliferated to 60% confluence.

[0094] Original method: iPSCs were subcultured using the classic subculture method for 5 consecutive generations. 24 hours after inoculation on the differentiation plate, the Rock inhibitor was removed and the cells were allowed to proliferate to 60% of the differentiation-initiating confluence.

[0095] This method: The subculture process adopts the screening subculture method of Example 1 for 5 consecutive generations, and retains the Rock inhibitor after inoculation and differentiation until the differentiation-initiating confluence reaches 60%.

[0096] After 13 days of differentiation in both groups, the proportion of ALB cells was analyzed by flow cytometry.

[0097] Flow cytometry results Figure 10 As shown in Figure 2, compared with the original method, this method improves ALB + The purity of hepatocytes was 15.9%.

[0098] Application 2: Mesoderm 2D derivatives

[0099] To verify whether this method is applicable to improving the differentiation efficiency of mesoderm 2D derivatives, we selected the iPSC-to-2D macrophage differentiation protocol for demonstration (Cao X, Yakala GK, van den Hil FE, et al. Differentiation and functional comparison of monocytes and macrophages fromhiPSCs with peripheral blood derivatives[J]. Stem cell reports, 2019, 12(6):1282-1297.). Based on the requirements of this method, differentiation was initiated after iPSCs proliferated to 100% differentiation confluence.

[0100] Original method: iPSCs were subcultured using the classic subculture method for 5 consecutive generations. 24 hours after inoculation on the differentiation plate, the Rock inhibitor was removed and the cells were allowed to proliferate to 100% differentiation-initiating confluence.

[0101] This method: The subculture process adopts the screening subculture method of Example 1 for 5 consecutive generations, and retains the Rock inhibitor after inoculation and differentiation to proliferate until the differentiation-initiating confluence reaches 100%.

[0102] After 20 days of differentiation, the CD14 + Cell proportion.

[0103] Flow cytometry results Figure 11 As shown in the figure, compared with the original method, this method improves CD14 + Macrophage purity 12.1%

[0104] Application 3: Ectodermal 2D derivatives

[0105] To verify whether this method is applicable to improving the differentiation efficiency of 2D ectoderm derivatives, we used an iPSC-to-2D GABAergic neuron differentiation protocol (Liu Y, Liu H, Sauvey C, et al. Directed differentiation of forebrain GABA interneurons from human pluripotent stem cells[J]. Nature protocols, 2013, 8(9): 1670-1679.). Based on the requirements of this method, differentiation was initiated after iPSCs proliferated to 80% confluence.

[0106] Original method: iPSCs were subcultured using the classic subculture method for 5 consecutive generations. 24 hours after inoculation on the differentiation plate, the Rock inhibitor was removed and the cells were allowed to proliferate to 80% of the differentiation-initiating confluence.

[0107] This method: The subculture process adopts the screening subculture method of Example 1 for 5 consecutive generations, and retains the Rock inhibitor after inoculation and differentiation to proliferate until the differentiation-initiating confluence reaches 80%.

[0108] After 80 days of differentiation, GABA expression in both groups was analyzed by flow cytometry. + Cell proportion.

[0109] Flow cytometry results Figure 12 As shown, compared with the original method, this method increases the purity of GABAergic neurons by 9.1%

[0110] Applications 1-3 demonstrated the differentiation of iPSCs into 2D derivatives (endodermal derivatives, mesoderm derivatives, and ectoderm derivatives), increasing differentiation efficiency by an average of 12.4%.

[0111] Application 4: Endoderm-derived 3D derivatives

[0112] To verify whether this method can improve the differentiation efficiency of endoderm-derived 3D derivatives, we selected the iPSC-to-3D colon organoid differentiation protocol for verification (Crespo M, Vilar E, Tsai SY, et al. Colonic organoids derived from human induced pluripotent stem cells for modeling colorectal cancer and drug testing[J]. Nature medicine, 2017, 23(7): 878-884.). Based on the requirements of this method, differentiation was initiated after iPSCs proliferated to 90% confluence.

[0113] Original method: iPSCs were subcultured using the classic subculture method for 5 consecutive generations. 24 hours after inoculation on the differentiation plate, the Rock inhibitor was removed and the cells were allowed to proliferate to 90% of the differentiation-initiating confluence.

[0114] This method: The subculture process adopts the screening subculture method of Example 1 for 5 consecutive generations, and retains the Rock inhibitor after inoculation and differentiation to proliferate until the differentiation-initiating confluence reaches 90%.

[0115] After 40 days of differentiation, CDX2 expression in both groups was analyzed by flow cytometry. + SATB2 + Cell proportion.

[0116] Flow cytometry results Figure 13 As shown in Figure 2, compared with the original method, this method improves CDX2 + SATB2 + The proportion of colon epithelium is 17.5%.

[0117] Application 5: Mesodermal-derived 3D derivatives

[0118] To verify whether this method can improve the differentiation efficiency of mesoderm-derived 3D derivatives, the iPSC differentiation protocol into 3D cardiac organoids was selected for verification (Prondzynski M, Berkson P, Trembley MA, et al. Efficient and reproducible generation of human iPSC-derived cardiomyocytes and cardiacorganoids in stirred suspension systems[J]. Nature Communications, 2024, 15(1): 5929.).

[0119] Original method: iPSCs were subcultured using the classic subculture method for 5 consecutive generations. 24 hours after inoculation on the differentiation plate, the Rock inhibitor was removed and the cells were allowed to proliferate to 80% of the differentiation-initiating confluence.

[0120] This method: The subculture process adopts the screening subculture method of Example 1 for 5 consecutive generations, and retains the Rock inhibitor after inoculation and differentiation to proliferate until the differentiation-initiating confluence reaches 80%.

[0121] After 15 days of differentiation in both groups, cTNTs were analyzed by flow cytometry. + Cell proportion.

[0122] Flow cytometry results Figure 14 The results showed that this method can increase cTNT + The myocardial induction efficiency was 13.9%.

[0123] Application 6: Ectodermal-derived 3D derivatives

[0124] To verify whether this method can improve the differentiation efficiency of ectoderm-derived 3D derivatives, the iPSC differentiation protocol into 3D brain organoids was selected for verification (González-Sastre R, Coronel R, Bernabeu-Zornoza A, et al. Efficient generation of human cerebral organoids directly from adherent cultures of pluripotent stem cells[J]. Journal of Tissue Engineering, 2024,15: 20417314231226027.).

[0125] Original method: iPSCs were subcultured using the classic subculture method for 5 consecutive generations. 24 hours after inoculation on the differentiation plate, the Rock inhibitor was removed and the cells were allowed to proliferate to 80% of the differentiation-initiating confluence.

[0126] This method: The subculture process adopts the screening subculture method of Example 1 for 5 consecutive generations, and retains the Rock inhibitor after inoculation and differentiation to proliferate until the differentiation-initiating confluence reaches 80%.

[0127] After 60 days of differentiation, SOX2 expression in both groups was analyzed by flow cytometry. + Cell proportion.

[0128] Flow cytometry results Figure 15 As shown, the results showed that this method can improve the induction efficiency of neuroepithelium by about 11.8%.

[0129] Applications 4-6 demonstrated iPSC differentiation into 3D derivatives (endodermal derivatives, mesoderm derivatives, and ectoderm derivatives), increasing differentiation efficiency by an average of 14.4%.

[0130] In summary, the method for improving iPSC differentiation efficiency described in the present invention combines screening-based subculture and Rock inhibitor-retention differentiation initiation, significantly improving differentiation efficiency. For example, the average induction efficiency of 2D derivatives increased by 12.4%, and the average efficiency of 3D derivatives increased by 14.4%, with differentiation efficiencies both exceeding 10%. It has universal applicability to iPSC derivatives, that is, it is applicable to the induced differentiation of 2D and 3D derivatives of iPSC endodermal, mesoderm, and ectoderm sources.

[0131] Table 1 List of reagents and consumables

[0132]

[0133] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for improving iPSC differentiation efficiency, characterized by: The method comprises the following steps: (1) Screening subculture: iPSCs with no discernible signs of differentiation and a double-positive rate of ≥85% for the pluripotency markers OCT4 and NANOG were selected. Conventionally passaged iPSCs were digested with Accutase for 2 minutes, and undifferentiated, completely dissociated cells were selectively recovered. These iPSCs were then passaged continuously for at least 5 generations, with each generation undergoing a 2-minute digestion and recovery of undifferentiated, completely dissociated cells. This gradually eliminated cells that were relatively insensitive to digestive enzymes, achieving the enrichment of enzyme-sensitive iPSCs. (2) Rock inhibitor retention differentiation initiation: The iPSCs collected at the end of step (1) were digested with Accutase for 5-10 min to completely dissociate them, and then inoculated into a culture medium containing 10 μM Rock inhibitor to proliferate and differentiate until differentiation was initiated.

2. The method for improving iPSC differentiation efficiency according to claim 1, wherein: In step (1), each time the cells are subcultured until the confluence reaches 70-80%, a short digestion is performed using digestive enzymes.

3. The method for improving iPSC differentiation efficiency according to claim 1, wherein: In step (2), the collected iPSCs were first restored to normal digestion conditions and the cells were collected, and then inoculated and proliferated at a density of 1-3×10 5 pieces / cm 2 .

4. The method for improving iPSC differentiation efficiency according to claim 1, wherein: In step (2), iPSCs are proliferated in a culture medium containing Rock inhibitor for 24-72 hours, with the culture medium being replaced every 24 hours until the starting confluence required by the corresponding differentiation protocol is met.

5. The method for improving iPSC differentiation efficiency according to claim 1, wherein: Before proceeding to step (1), bright field morphology and pluripotency marker quality testing are required to select iPSCs of qualified quality for the experiment.

6. The method for improving iPSC differentiation efficiency according to claim 5, wherein: The quality inspection standards are: Ⅰ. Microscopic examination using an optical microscope: if >90% of the cells exhibit rounded, tightly connected, typical iPSC morphology, and <10% of the cells show signs of differentiation, the cells are considered to have passed morphological examination. II. Perform immunofluorescence staining of candidate iPSCs using antibodies against the pluripotency markers OCT4 and NANOG. If the double-positive rate is ≥85%, the candidate iPSCs are considered to have passed the pluripotency test. If all the above are inspected, the quality is considered qualified.

7. The method for improving iPSC differentiation efficiency according to claim 1, wherein: Rock inhibitors include Y-27632 and Thiazovivin.

8. A use of the method for improving iPSC differentiation efficiency according to any one of claims 1 to 7, characterized in that: This method is applied to improve the differentiation efficiency of 2D derivative differentiation and 3D derivative differentiation.

9. Use of the method for improving iPSC differentiation efficiency according to claim 8, characterized in that: 2D derivative differentiation includes endoderm 2D derivative differentiation, mesoderm 2D derivative differentiation and ectoderm 2D derivative differentiation, and the average differentiation efficiency of 2D derivative differentiation is increased by more than 10%.

10. Use of the method for improving iPSC differentiation efficiency according to claim 8, characterized in that: 3D derivative differentiation includes endoderm 3D derivative differentiation, mesoderm 3D derivative differentiation and ectoderm 3D derivative differentiation, and the average differentiation efficiency of 3D derivative differentiation is increased by more than 10%.

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