IPSC differentiation efficiency improving method and application

Abstract

The invention provides an iPSC (induced pluripotent stem cell) differentiation efficiency improvement method and application, and the method realizes the extensive improvement of the induction efficiency of different 2D / 3D (two-dimensional / three-dimensional) derivatives of an endoderm source, an ectoderm source and a mesoderm source by combining screening type subculture and Rock inhibitor retention type differentiation initiation.

Description

Technical Field

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

[0002] Since the Japanese team of S Yamanaka first reprogrammed somatic cells into induced pluripotent stem cells (iPSCs) by introducing four retroviral factors in 2006, in the past 20 years, based on in vitro induction techniques of developmental signals, researchers from various countries have successively established methods for differentiating iPSCs into different 2D and 3D derivatives of the human body; such as differentiating into 2D cells such as hepatocytes, cardiomyocytes, pancreatic islet cells, etc., and various 3D organoids such as the liver, lung, and kidney. Compared with traditional passaged cells / tissues, this new technology not only solves the problem of source restriction at one stroke, but also reproduces the key functional characteristics of the corresponding part in vivo in vitro. Therefore, it has played a broad application value in multiple fields such as disease simulation, drug screening, cell therapy, and organ repair.

[0003] With the advancement of the industrialization process, the problems of low yield and high preparation cost caused by insufficient induction efficiency have become the main bottleneck restricting large-scale transformation and urgently need to be overcome. Currently, the means to improve the induction efficiency mainly focus on adjusting technical parameters during the differentiation process. For example, in the differentiation of 2D hepatocytes, Y Nagamoto et al. increased the induction efficiency and achieved an increase in the purity of ALB + hepatocytes by overexpressing hepatocyte nuclear factor HNF1a. Another example is that in the induction and differentiation of 3D lung organoids, the HW Snoeck team enhanced the anterior induction efficiency by adding a Wnt signal inhibition step during the anterior induction stage, resulting in a significant increase in the yield of lung progenitor cells (NKX2.1 + ), and thus improving 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 optimizations such as acclimation culture before differentiation and differentiation seeding are still an overlooked aspect; there are few reports on improving the induction efficiency based on the improvement of iPSC culture technology. Summary of the Invention

[0005] In view of this, the present invention aims to propose a method for improving the differentiation efficiency of iPSCs and its application. This method is a pre-differentiation condition optimization method, which combines screening-based passage culture and Rock inhibitor retention-based differentiation initiation to achieve a general 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 realized as follows: A method for improving the differentiation efficiency of iPSCs, the method comprising the following steps: (1) Screening-based subculture: Use digestive enzymes to perform short-term digestion of routinely passaged iPSCs for 1.5 - 2 minutes, selectively recover undifferentiated cells, and then continuously passage them to at least 5 generations. And short-term digestion and undifferentiated cell recovery are performed for each generation, so as to gradually screen out cells that are relatively insensitive to digestive enzymes, realizing the enrichment of enzyme-sensitive iPSCs, and these enzyme-sensitive iPSCs have higher differentiation efficiency; (2) Rock inhibitor retention-based differentiation initiation: Inoculate the iPSCs collected in the last step (1) in a medium containing a Rock inhibitor to proliferate and differentiate until differentiation is initiated.

[0007] In some embodiments, in step (1), each time the subculture is carried out until the confluence reaches 70 - 80%, short-term digestion is performed with digestive enzymes.

[0008] Among them, the digestive enzyme is preheated to 37 °C for use.

[0009] In some embodiments, in step (2), the collected iPSCs are first restored to the conventional digestion conditions and the cells are collected, and then inoculated for proliferation. The inoculation density is 1 - 3×10 5 cells / cm 2 .

[0010] The conventional digestion conditions are: the digestive enzyme digests for 5 - 10 minutes until all cells are dissociated.

[0011] In some embodiments, in step (2), the iPSCs proliferate in a medium containing a Rock inhibitor for 24 - 72 hours, and the medium is changed every 24 hours until the initiation confluence required by the corresponding differentiation protocol is met.

[0012] In some embodiments, before performing step (1), bright-field morphology and pluripotency marker quality verification are required, and iPSCs with qualified quality are selected for the experiment.

[0013] Furthermore, the quality verification criteria are: Ⅰ. Use an optical microscope for microscopy. If more than 90% of the cells show the typical morphological characteristics of round and tightly connected iPSCs, and less than 10% of the cells show differentiation traces, it is regarded as passing the morphological inspection; Ⅱ. Use pluripotency markers OCT4 and NANOG antibodies to perform immunofluorescence staining on candidate iPSCs; if the double-positive rate ≥ 85%, it is regarded as passing the pluripotency inspection; If all of the above pass the inspection, it is regarded as having qualified quality.

[0014] In some embodiments, the Rock inhibitor includes Y-27632, Thiazovivin, and the concentration of the Rock inhibitor is 2-50 μM.

[0015] Among them, the Rock inhibitor includes but is not limited to Y-27632, Thiazovivin, and can also be other Rock inhibitors that can achieve the purpose of the present invention. Only limited examples are listed here; preferably, the Rock inhibitor can be Y-27632.

[0016] Preferably, the concentration of Y-27632 is 10-50 μM. Non-limiting examples can be 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and more preferably 10 μM; The concentration of Thiazovivin is 2-10 μM. Non-limiting examples can be 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, 10 μM.

[0017] The present invention also provides an application of the above-mentioned method for improving the differentiation efficiency of iPSCs, and this method is applied to improving the differentiation efficiency of 2D derivative differentiation and 3D derivative differentiation.

[0018] 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 has increased by more than 10%.

[0019] 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 has increased by more than 10%.

[0020] Compared with the prior art, the method and application for improving the differentiation efficiency of iPSCs according to the present invention have the following advantages: (1) The method for improving the differentiation efficiency of iPSCs according to 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 has increased by 12.4%, and the average efficiency of 3D derivatives has increased by 14.4%. The differentiation efficiency has increased by more than 10% in both cases; it has broad applicability to iPSC derivatives, that is, it is applicable to the induced differentiation of 2D and 3D derivatives from iPSC endoderm sources, mesoderm sources, and ectoderm sources.

[0021] (2) In the screening-based subculture of the present invention, through continuous short-term subculture, cells that are relatively insensitive to digestive enzymes are gradually screened out to achieve the enrichment of enzyme-sensitive iPSCs. Studies have shown that these enzyme-sensitive iPSCs have higher differentiation efficiency.

[0022] (3) In the Rock inhibitor-retained differentiation initiation of the present invention, the Rock inhibitor was originally used for iPSC seeding, which can increase the adhesion rate and then improve the cell viability, and was removed on the day after seeding. It was found in the present invention that the presence of the Rock inhibitor can keep the iPSC cells maintaining a small distance from each other, changing from the previous classical clone-like tight connection state to a small separated state, so that the cells can synchronously respond to the induction factors, thereby improving the differentiation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments and descriptions thereof of the present invention are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings: Figure 1 is a flow chart of the method for improving the differentiation efficiency of iPSC; Figure 2 is the immunofluorescence staining assay of iPSC morphology and pluripotency markers, scale bar = 200 μm; Figure 3 is the bright-field morphology diagram of iPSC after endoderm differentiation for 24 h after single classical passage and screening passage respectively; scale bar = 200 μm; Figure 4 is the quantitative analysis comparison of the cell confluence of the cells after one passage by the two methods; n = 4 biological replicates; calculated using Image J software, cell confluence (%) = (total frame area - well plate bare area) / total frame area × 100%, n = 3 biological replicates; **, P < 0.01; Figure 5 is the bright-field morphology diagram and the quantitative result of cell confluence of iPSC after endoderm differentiation for 24 h after continuous classical passage and screening passage; scale bar = 200 μm; Figure 6 is the quantitative analysis comparison result of the cell confluence of iPSC after endoderm differentiation for 24 h after continuous classical passage and screening passage; n = 4 biological replicates; **, P < 0.01; ***, P < 0.001; Figure 7 is the SOX17 + cell ratio detected by flow cytometry after 72 h of endoderm differentiation of iPSC after continuous screening passage, n = 3 biological replicates; Figure 8 is the SOX17 + cell ratio detected by flow cytometry after 72 h of endoderm differentiation of iPSC after 5 classical passages and screening passages respectively; n = 10 biological replicates; ***, P < 0.001; SOX17, endoderm marker; Figure 9 Flow cytometry result comparison chart for three differentiation methods; n = 10 biological replicates; **, P < 0.01; ***, P < 0.001; Figure 10 Comparison of the differentiation efficiency of iPSCs into 2D hepatocytes, ALB, a hepatocyte marker; n = 10 biological replicates; ***, P < 0.001; Figure 11 Comparison of the differentiation efficiency of iPSCs into 2D macrophages, CD14, a macrophage-specific marker; n = 10 biological replicates; ***, P < 0.001; 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; Figure 13 Comparison of the differentiation efficiency of iPSCs into 3D intestinal organoids; CDX2, a broad-spectrum marker for intestinal epithelium; SATB2, a colon epithelium-specific marker; n = 10 biological replicates; **, P < 0.001; Figure 14 Comparison of the differentiation efficiency of iPSCs into 3D heart organoids, cTNT, a myocardial-specific marker; n = 10 biological replicates; ***, P < 0.001; Figure 15 Comparison of the differentiation efficiency of iPSCs into 3D brain organoids, SOX2, a neuroepithelial marker; n = 10 biological replicates; **, P < 0.01. Detailed implementation manners

[0024] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other.

[0025] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0026] This method is applicable to the classical iPSC culture system based on mTeSR1 / Matrigel. Some of the reagents used in the present invention are shown in Table 1.

[0027] Example 1 iPSC differentiation As Figure 1 shown, the method for improving the differentiation efficiency of iPSCs includes the following steps: 1. Select iPSCs with qualified quality: Take the candidate iPSCs and perform quality verification through bright-field morphology and pluripotency markers: 1) Use an optical microscope for microscopy. If >90% of the cells show the typical morphological characteristics of round and tightly connected iPSCs and <10% of the cells show signs of differentiation, it is considered to pass the morphological inspection.

[0028] 2) Perform immunofluorescence staining of the candidate iPSCs with the pluripotency markers OCT4 and NANOG antibodies; if the double-positive rate ≥85%, it is considered to pass the pluripotency inspection.

[0029] If all of the above pass the inspection, it is considered to have qualified quality and can be selected for subsequent operations.

[0030] The inspection results are as Figure 2 shown. The upper figure is the morphology of iPSCs. It can be seen that there are no distinguishable signs of differentiation, and all cells show the typical morphological characteristics of round and tightly connected iPSCs; the 4 lower figures are the immunofluorescence staining inspection of pluripotency markers, and the double-positive rate of the pluripotency markers OCT4 and NANOG is 86%.

[0031] Therefore, it is determined that the quality is qualified, and it is selected for screening and passage.

[0032] 2. Screening-based passage culture: Take the iPSCs with qualified quality and continuously screen for sensitive iPSCs by short-term digestion: 1) Short-term digestion: When reaching the conventional passage confluence (70 - 80%), use Accutase cell dissociation enzyme preheated to 37°C and strictly control the time for 2 minutes for digestion. Thereafter, only collect the undifferentiated, completely dissociated iPSCs that are considered more sensitive to the digestion enzyme at this time period for continuous amplification; the remaining undissociated cells are considered relatively insensitive or less sensitive and are discarded.

[0033] 2) Continuous screening: After the cells to be collected reach 70 - 80% confluence again, repeat step 1) until continuous passage for 5 times, which is considered the completion of the screening of sensitive iPSCs and enter the differentiation inoculation.

[0034] 3. Rock inhibitor-retained differentiation initiation Take the sensitive iPSCs and inoculate them into the differentiation plate, and retain the Rock inhibitor during the proliferation process until the differentiation is initiated: 1) Complete collection of iPSCs: At the corresponding passage of the differentiation inoculation, restore the conventional digestion conditions, that is, Accutase digestion for 5 - 10 minutes to completely dissociate, and collect all cells. The specific digestion time varies depending on the iPSC cell line.

[0035] 2) Differentiated plate seeding: After resuspending with the mTeSR1 medium containing 10 μM Rock inhibitor, seed at an inoculation density of 1×10 5 cells / cm 2 . Specifically, the Rock inhibitor can be Y-27632.

[0036] 3) Initiate differentiation: Wait for proliferation for 24 - 72 h, and replace the new medium containing 10 μM Rock inhibitor every 24 h until the starting confluence required by the corresponding differentiation protocol is met.

[0037] Among them, the proliferation time is based on the target starting confluence required by different differentiation protocols, and different proliferation times are required.

[0038] Verification 1 The role of screening passage To explore the role of screening passage, in parallel, a classical passage group was set up. First, the directed differentiation of iPSC into endodermal cells was used for verification (D'Amour K A, Agulnick A D, Eliazer S, et al. Efficient differentiation of human embryonic stem cells to definitive endoderm[J]. Nature biotechnology, 2005, 23(12): 1534 - 1541.). According to the requirements of this differentiation method, the iPSC of both passage methods grew to 90% confluence to initiate differentiation, and the differentiation process was analyzed.

[0039] Classical passage: In the digestion step, Accutase was used for digestion for 5 - 10 min, and all cells were collected and continued to be cultured.

[0040] Screening passage: In the digestion step, only digest for 2 min, and collect the corresponding dissociated cells and continue to be cultured.

[0041] The analysis results are as Figures 3 - 8 shown: As Figures 3 - 4 For the analysis after a single classical passage and screening passage, compared with the classical passage, the iPSC obtained by short-time digestion in the screening passage had a lower cell confluence after 24 h (average 83.3% vs 70.9%). This result suggests that these iPSC that are more sensitive to Accutase dispersing enzyme obtained by short-time digestion screening are also more sensitive to the cytokine Activin A.

[0042] As Figures 5 - 8For continuous classical passage and analysis after screening passage, different from classical passage, continuous screening passage gradually increases the differentiation sensitivity and remains stable after 5 passages ( Figures 5 - 6 ). Flow cytometry analysis after 72 hours of differentiation showed that the increased sensitivity caused by continuous screening passage could be converted into an improvement in the endoderm differentiation efficiency, and there was a positive correlation between the two overall ( Figures 6 - 7 ). Finally, the comparison results based on 10 induced differentiation experiments showed that after 5 passages, screening passage increased the endoderm cell differentiation efficiency by about 7.2% ( Figure 8 ).

[0043] Verification of the effect of Rock inhibitor 2 Using the above-mentioned endoderm cell differentiation method, the retention effect of the Rock inhibitor was investigated. Three groups were set up in parallel: Rock inhibitor withdrawal differentiation of iPSCs obtained by the classical passage method, Rock inhibitor retention differentiation of iPSCs obtained by the classical passage method, and Rock inhibitor retention differentiation of iPSCs obtained by the screening passage method.

[0044] Classical passage method: Digest with Accutase for 5 - 10 minutes, collect all cells and continue culturing; after continuous passage 5 times, the obtained iPSCs.

[0045] Screening passage method: Digest for 2 minutes, only collect the corresponding detached cells and continue culturing; after continuous passage 5 times, the obtained iPSCs.

[0046] Rock-i (Rock inhibitor) withdrawal: After inoculating the differentiation plate for 24 hours, remove the Rock inhibitor and continue culturing until 90% confluence is reached at the start.

[0047] Rock-i (Rock inhibitor) retention: Retain the Rock inhibitor after inoculating the differentiation plate and continue culturing until 90% confluence is reached at the start.

[0048] The Rock-i inhibitor selected was 10 μM Y-27632.

[0049] The flow cytometry results after differentiation are as Figure 9 shown. In the case of iPSCs using the classical passage method, Rock inhibitor retention differentiation could increase the endoderm differentiation efficiency by 5.2%; while combined with screening passage, the differentiation efficiency would be increased by 11.9%. This shows that on the basis of screening passage, the retention of the Rock inhibitor further improves the differentiation efficiency of iPSCs into endoderm cells.

[0050] The following will use the evidence of different applications to clarify the general improvement effect of this method on the differentiation efficiency of 2D and 3D derivatives from iPSCs.

[0051] Application 1: Endodermal 2D derivatives To verify the improvement effect of this method on the differentiation efficiency of endodermal 2D derivatives, it was verified based on a protocol for the directed differentiation of iPSCs into 2D hepatocytes (Sullivan G J, Hay D C, Park I H, et al. Generation of functional human hepatic endoderm from human induced pluripotent stem cells[J]. Hepatology, 2010, 51(1): 329-335.). According to the requirements of this method, after the iPSCs proliferated to 60% confluence for differentiation, differentiation was initiated.

[0052] Original method: During the subculture process of iPSCs, they were continuously cultured for 5 generations using the classical subculture method. After inoculating the differentiation plate for 24 hours, the Rock inhibitor was removed, and they were allowed to proliferate to 60% confluence for the initiation of differentiation.

[0053] This method: During the subculture process, they were continuously cultured for 5 generations using the screening subculture method of Example 1, and the Rock inhibitor was retained after inoculation for differentiation until the confluence for the initiation of differentiation reached 60%.

[0054] After 13 days of differentiation in both groups, flow cytometry was used to analyze the proportion of ALB cells.

[0055] The results of flow cytometry are as Figure 10 shown. Compared with the original method, this method increased the purity of ALB + hepatocytes by 15.9%.

[0056] Application 2: Mesodermal 2D derivatives To verify whether this method is applicable to the improvement of the differentiation efficiency of mesodermal 2D derivatives, a protocol for the directed differentiation of iPSCs into 2D macrophages was selected for proof (Cao X, Yakala G K, van den Hil F E, et al. Differentiation and functional comparison of monocytes and macrophages from hiPSCs with peripheral blood derivatives[J]. Stem cell reports, 2019, 12(6): 1282-1297.). According to the requirements of this method, after the iPSCs proliferated to 100% confluence for differentiation, differentiation was initiated.

[0057] Original method: In the process of iPSC subculture, the classical subculture method was used for continuous culture for 5 generations. After 24 hours of inoculating the differentiation plate, the Rock inhibitor was removed, and the cells were proliferated until the confluence of differentiation initiation reached 100%.

[0058] This method: In the process of subculture, the screening subculture method of Example 1 was used for continuous culture for 5 generations. After inoculating for differentiation, the Rock inhibitor was retained for proliferation until the confluence of differentiation initiation reached 100%.

[0059] After both groups were differentiated for 20 days, flow cytometry was used to analyze the proportion of CD14 + cells.

[0060] The results of flow cytometry are as Figure 11 shown. Compared with the original method, this method increased the purity of CD14 + macrophages by 12.1% Application 3 2D derivatives of the three germ layers To verify whether this method is applicable to improving the differentiation efficiency of 2D derivatives of the three germ layers, an iPSC to 2D GABAergic neuron differentiation protocol was used for proof (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, after the iPSCs were proliferated to 80% confluence of differentiation, differentiation was initiated.

[0061] Original method: In the process of iPSC subculture, the classical subculture method was used for continuous culture for 5 generations. After 24 hours of inoculating the differentiation plate, the Rock inhibitor was removed, and the cells were proliferated until the confluence of differentiation initiation reached 80%.

[0062] This method: In the process of subculture, the screening subculture method of Example 1 was used for continuous culture for 5 generations. After inoculating for differentiation, the Rock inhibitor was retained for proliferation until the confluence of differentiation initiation reached 80%.

[0063] After both groups were differentiated for 80 days, flow cytometry was used to analyze the proportion of GABA + cells.

[0064] The results of flow cytometry are as Figure 12 shown. Compared with the original method, this method increased the purity of GABAergic neurons by 9.1% Application 1-3 demonstrated the differentiation of iPSCs into 2D derivatives (endoderm derivatives, mesoderm derivatives, and ectoderm derivatives), with an average increase in differentiation efficiency of 12.4%.

[0065] Application 4 Endoderm-derived 3D derivatives To verify whether this method can improve the differentiation efficiency of endoderm-derived 3D derivatives, the differentiation protocol of iPSC into 3D colonic organoids was selected for verification (Crespo M, Vilar E, Tsai S Y, 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, after the iPSC proliferated to 90% confluence for differentiation, differentiation was initiated.

[0066] Original method: During the subculture process of iPSC, continuous subculture was performed for 5 generations using the classical subculture method. After 24 hours of inoculation on the differentiation plate, the Rock inhibitor was removed, and proliferation was carried out until 90% confluence for the start of differentiation.

[0067] This method: During the subculture process, continuous subculture was performed for 5 generations using the screening subculture method of Example 1, and the Rock inhibitor was retained for proliferation after inoculation for differentiation until 90% confluence for the start of differentiation.

[0068] After 40 days of differentiation in both groups, flow cytometry was used to analyze the proportion of CDX2 + SATB2 + positive cells.

[0069] The results of flow cytometry are as Figure 13 shown. Compared with the original method, this method increased the proportion of CDX2 + SATB2 + positive colonic epithelium by 17.5%.

[0070] Application 5 Mesoderm-derived 3D derivatives To verify whether this method can improve the differentiation efficiency of mesoderm-derived 3D derivatives, the differentiation protocol of iPSC into 3D heart organoids was selected for verification (Prondzynski M, Berkson P, Trembley M A, et al. Efficient and reproducible generation of human iPSC-derived cardiomyocytes and cardiac organoids in stirred suspension systems[J]. Nature Communications, 2024, 15(1): 5929.).

[0071] Original method: During the subculture process of iPSC, it was continuously cultured for 5 generations using the classical subculture method. After 24 hours of inoculating the differentiation plate, the Rock inhibitor was removed, and it was proliferated to 80% confluence for differentiation initiation.

[0072] This method: During the subculture process, it was continuously cultured for 5 generations using the screening subculture method of Example 1, and the Rock inhibitor was retained for proliferation after inoculation until 80% confluence for differentiation initiation was reached.

[0073] After 15 days of differentiation in both groups, flow cytometry was used to analyze the proportion of cTNT + cells.

[0074] The results of flow cytometry are as Figure 14 shown, and the results show that this method can improve the cTNT + cardiomyocyte induction efficiency by 13.9%.

[0075] Application of 6 ectoderm-derived 3D derivatives To verify whether this method can improve the differentiation efficiency of ectoderm-derived 3D derivatives, the differentiation protocol of iPSC 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.).

[0076] Original method: In the process of iPSC passage culture, the classical passage method was used for continuous culture for 5 generations. After 24 hours of inoculating the differentiation plate, the Rock inhibitor was removed, and the cells were proliferated to 80% of the confluence for differentiation initiation.

[0077] This method: In the process of passage culture, the screening passage method of Example 1 was used for continuous culture for 5 generations. After inoculation and differentiation, the Rock inhibitor was retained for proliferation until 80% of the confluence for differentiation initiation was reached.

[0078] After 60 days of differentiation in both groups, flow cytometry was used to analyze the proportion of SOX2 + cells.

[0079] The results of flow cytometry are as Figure 15 shown. The results indicate that this method can improve the neuroepithelial induction efficiency by about 11.8%.

[0080] Examples 4 - 6 demonstrate the differentiation of iPSCs into 3D derivatives (endoderm derivatives, mesoderm derivatives, and ectoderm derivatives), with an average increase in differentiation efficiency of 14.4%.

[0081] In summary, the method for improving the differentiation efficiency of iPSCs according to the present invention combines screening passage culture and Rock inhibitor - retained differentiation initiation, significantly improving the differentiation efficiency. For example, the average induction efficiency of 2D derivatives is increased by 12.4%, and the average efficiency of 3D derivatives is increased by 14.4%. The differentiation efficiency is increased by more than 10% in both cases. It has broad applicability to iPSC derivatives, that is, it is applicable to the induced differentiation of 2D and 3D derivatives from endoderm - derived, mesoderm - derived, and ectoderm - derived iPSCs.

[0082] Table 1 List of reagents and consumables

[0083] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for improving the differentiation efficiency of iPSCs, characterized in that: The method comprises the following steps: (1) Screening-based subculture: The iPSCs of conventional subculture are digested briefly with a digestive enzyme for 1.5 - 2 min, undifferentiated cells are selectively recovered, and then continuously subcultured to at least 5 generations. Short-time digestion and undifferentiated cell recovery are carried out for each generation, thereby gradually screening out cells that are relatively insensitive to the digestive enzyme and achieving enrichment of enzyme-sensitive iPSCs; (2) Initiation of differentiation with Rock inhibitor retention: The iPSCs collected at the end of step (1) are inoculated in a medium containing a Rock inhibitor to proliferate and differentiate until the differentiation is initiated.

2. The method for improving the differentiation efficiency of iPSCs according to claim 1, characterized in that: In step (1), when the confluence reaches 70 - 80% during each subculture, short-time digestion is carried out with a digestive enzyme.

3. The method for improving the differentiation efficiency of iPSCs according to claim 1, characterized in that: In step (2), the collected iPSCs are first restored to normal digestion conditions and the cells are collected, and then inoculated for proliferation, with an inoculation density of 1-3×10 5 Pieces / cm 2 .

4. The method for improving the differentiation efficiency of iPSCs according to claim 1, characterized in that: In step (2), the iPSCs proliferate in a medium containing a Rock inhibitor for 24 - 72 h, and the medium is changed every 24 h until the initiation confluence required by the corresponding differentiation protocol is met.

5. The method for improving the differentiation efficiency of iPSCs according to claim 1, characterized in that: Before step (1) is carried out, bright-field morphology and pluripotency marker quality verification are required, and iPSCs with qualified quality are selected for the experiment.

6. The method for improving the differentiation efficiency of iPSCs according to claim 5, characterized in that: The criteria for quality verification are as follows: Ⅰ. Using an optical microscope for microscopic examination, if more than 90% of the cells show the typical morphological characteristics of round and tightly connected iPSCs, and less than 10% of the cells show differentiation traces, it is regarded as passing the morphological examination; Ⅱ. Immunofluorescent staining of candidate iPSCs is carried out using pluripotency markers OCT4 and NANOG antibodies; if the double-positive rate ≥ 85%, it is regarded as passing the pluripotency examination; If both of the above pass the examination, it is regarded as having qualified quality.

7. The method for improving the differentiation efficiency of iPSCs according to claim 1, characterized in that: The Rock inhibitor includes Y-27632, Thiazovivin, and the concentration of the Rock inhibitor is 2 - 50 μM.

8. An application of the method for improving the differentiation efficiency of iPSCs according to any one of claims 1-7, characterized in that: This method is applied to improving the differentiation efficiency of 2D derivatives differentiation and 3D derivatives differentiation.

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

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

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