Method for induced differentiation of human pluripotent stem cells into pancreatic precursor cells

Abstract

The invention relates to a method for induced differentiation of human pluripotent stem cells into pancreatic precursor cells, compared with a traditional induced differentiation process, according to the technical scheme provided by the invention, the process of consuming more than 30 days and needing 6-7 stages is shortened to 5 stages of only 19 days, and the proportion of beta-like cells can be significantly increased to 60-70%, so that the method has the advantages that the cost is reduced, and the method is suitable for large-scale popularization and application. According to the present invention, the hyperglycemia symptom in the diabetic mouse model is effectively improved, and the single cell transcriptome detection is performed on the graft to discover that 60-70% of beta cells can be detected, such that the cell state is more mature compared with the pre-transplantation cell state. In addition, the invention also discloses a method for evaluating the cell quality, which is used for measuring the quality of in-vitro induced cells. The method does not depend on the traditional detection based on a small number of specific genes and protein expression any more, but utilizes a unicellular omics technology to qualitatively evaluate the characteristics and differentiation efficiency of the induced cells based on three modules of a gene co-expression network, and a brand new evaluation standard is provided for the field.

Description

[0001] This application is a divisional application. The filing date of the original application is April 10, 2024, the application number is 202410431275.4, and the invention title is "A method for inducing differentiation of human pluripotent stem cells into pancreatic progenitor cells". Technical Field

[0002] The present invention relates to the field of biotechnology. Specifically, the present invention relates to a method for inducing differentiation of human pluripotent stem cells into pancreatic progenitor cells. Background Art

[0003] Diabetes, a global chronic disease marked by persistent hyperglycemia, is mainly caused by insufficient insulin secretion or resistance to insulin. The long-term hyperglycemic environment may lead to damage and dysfunction of various tissues including the heart, blood vessels, kidneys, eyes, and nerves. Current treatment strategies, such as exogenous insulin injection and islet cell transplantation, can alleviate diabetes symptoms, but suffer from problems such as insufficient accuracy, hypoglycemia risk, and shortage of donor cells. With the development of stem cell technology, especially the strategy of obtaining β-like cells with insulin-secreting ability and islet-like structures from human pluripotent stem cells (such as human embryonic stem cells and human induced pluripotent stem cells) through in vitro directed differentiation technology, new treatment possibilities for diabetes have emerged.

[0004] Starting from human pluripotent stem cells (including human embryonic stem cells and human induced pluripotent stem cells), the development process of the pancreatic lineage in vivo is mimicked by adding small molecules and recombinant proteins to regulate signaling pathways, and finally islet-like tissues containing insulin-secreting β-like cells are obtained. It is generally believed that this process mainly involves stepwise induction into definitive endoderm (DE), primitive gut tube (PGT), posterior foregut (PF), pancreatic endoderm (PE) or pancreatic progenitor (PP), and endocrine progenitor (EP), and finally islet-like tissues containing β-like cells are obtained. Among them, the induction of high-quality pancreatic progenitor cells is crucial for the differentiation efficiency and function of subsequent endocrine cells. Due to the limited current understanding of human pancreatic development, the clues for directed differentiation mainly come from animal models, especially mice. Although there are corresponding specific genes or proteins for the above stages to judge the differentiation efficiency, there is a lack of a comprehensive evaluation method for the differentiation path and cell state in the whole process, and the similarities and differences with the real development process in the human body are still unclear. Therefore, it is extremely important to guide the differentiation of human pluripotent stem cells into high-quality pancreatic progenitor cells according to the differentiation path and regulatory network of the human pancreatic lineage, as well as the conservation and specificity of the regulation of pancreatic lineage development among species.

[0005] Current existing protocols have obtained, to a certain extent, induced cells expressing the PP cell characteristic genes PDX1 and NKX6-1 by gradually using different combinations of small molecule compounds and treatment sequences, as well as different culture durations, and on this basis, a certain proportion of β-like cells with certain functions have been obtained. However, at the same time, a relatively high proportion of multi-hormone cells (co-expression of insulin and glucagon) or α-like cells (secreting glucagon) will also be generated. The main reasons for the limited differentiation efficiency and purity of the current induction protocols are the incomplete understanding of the human pancreatic lineage differentiation process, the lack of a systematic evaluation of the in vitro differentiation path and induced cell state based on the in vivo differentiation process at the single-cell level, and the poor early differentiation process and PP cell state may be one of the reasons for subsequent differentiation problems. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention deeply analyzes the process of mouse and human pancreas development and finds that the developmental path is conserved among species but has certain specificity in the regulatory network. From the perspective of the developmental path, pancreatic progenitor cells (PP) have two origins, the ventral and dorsal pancreatic endoderm (VPE and DPE), and are derived from DE cells through AL cells (AL-P) with a pancreatic differentiation fate and MG cells (MG-P) with a pancreatic differentiation fate, respectively. Comparing the developmental paths of PP cells in humans and mice, it is found that in humans, the ventral path (AL-P and VPE) has a higher similarity to PP cells than the dorsal path (MG-P and DPE), indicating a higher differentiation efficiency, while this phenomenon is not obvious in mice. Through gene co-expression network analysis, it is further confirmed that the fine differentiation process from DE to the endocrine lineage includes three key gene modules, and PP cells are in a key position connecting the first two modules.

[0007] Subsequently, the inventors conducted a global single-cell level analysis of the existing induction protocol, identifying the cell properties, differentiation paths, and regulatory networks of the existing protocol. By comparing with the in vivo cell differentiation process, the inventors found two main differences from in vivo: one is that a stable gene co-expression network similar to in vivo development has not been established. The current in vitro differentiation process from DE to endocrine cells is divided into four gene modules, rather than three modules in vivo, and the early gene modules have not been stably established. The second is that the formation of PP cells in the existing protocol fails to mimic the efficient process of the ventral differentiation path in the human body, resulting in insufficient similarity between the transitional state transformed into PP cells and AL-P cells in the human body.

[0008] In view of this, the present invention proposes an improved method: by prolonging the induction duration of the S2 stage, promoting the formation of the early gene network, and imitating the natural transformation process of the ventral pancreatic endoderm to PP cells in human embryonic development (DE–AL-P–VPE–PP), the TGF-β Nodal pathway (adding Activin A) and the WNT pathway (adding CHIR-99021) are activated by adding specific signaling substances to promote the generation of cells in the AL-P state. On this basis, the S3 and S4 induction periods of the control method can be degenerate, and the usage concentration of FGF10 can be reduced to improve the quality of PP cells. Without changing the S5 and S6 induction protocols of the control method, the proportion of β-like cells can still be significantly increased, and the proportion of multi-hormone or α-like cells can be reduced. Analyzing the islet-like cells transplanted into animals at different induction times shows that the last induction stage (S7) of the control method can be omitted in this process, shortening the entire induction process to 5 stages in 19 days, and the transplanted islet-like cells can effectively maintain blood glucose stability.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] In the first aspect of the present invention, there is provided a method for inducing the differentiation of human pluripotent stem cells into pancreatic progenitor cells, the method comprising the following steps:

[0011] S1: Activate the TGF-β Nodal pathway and the WNT pathway simultaneously;

[0012] S2: Activate the FGF pathway, the TGF-β Nodal pathway, the WNT pathway and add vitamin C at a final concentration of 0.25 mM;

[0013] S3: Activate the FGF pathway, inhibit the SHH pathway, activate the Retinoic acid (RA) pathway, inhibit the BMP pathway, activate PKC and add vitamin C at a final concentration of 0.25 mM;

[0014] S4: Inhibit the SHH pathway, activate the Retinoic acid (RA) pathway, inhibit the BMP pathway, inhibit the TGF-βRI kinase and add thyroxine T3 at a final concentration of 1 μM;

[0015] S5: Inhibit the BMP pathway, activate the Notch pathway, inhibit the TGF-βRI kinase and add thyroxine T3 at a final concentration of 1 μM.

[0016] In one embodiment, the steps of the method are specifically as follows:

[0017] Culture human pluripotent stem cells in mTeSR1 pluripotent stem cell medium supplemented with a final concentration of 10 μM Y-27632, and initiate induced differentiation after 24 hours:

[0018] S1 lasts for 2 days: Add Activin A and CHIR-99021 at a final concentration of 100 ng / mL to the medium, with a final concentration of 3 μM on the first day and 0.3 μM on the second day;

[0019] S2 lasts for 4 days: Add FGF10 at a final concentration of 50 ng / mL, Activin A at 10 ng / mL, CHIR-99021 at 0.3 μM and vitamin C at 0.25 mM to the medium;

[0020] S3 lasts for 3 days: Add FGF10 at a final concentration of 2 ng / mL, 0.25 μM SANT-1, 1 μM RA, 100 nM LDN-193189, 200 nM TPPB and vitamin C at 0.25 mM to the medium;

[0021] S4 lasts for 3 days: Add 0.25 μM SANT-1, 0.05 μM RA, 100 nM LDN-193189, 10 μM ALK5i II, 1 μM thyroxine T3 to the medium;

[0022] S5 lasted for 7 days: Add 100 nM LDN-193189, 100 nM GSIXX, 10 μM ALK5iII, and 1 μM thyroxine T3 at the final concentration to the culture medium.

[0023] Among them, the basal medium used in S1 and S2 is MCDB 131 medium, additionally adding 1.5 g / L NaHCO3, 1×GlutaMAX, 10 mM D-glucose, 0.5% BSA, and 100 U / mL penicillin-streptomycin at the final concentration.

[0024] The basal medium used in S3 is: MCDB 131 medium, additionally adding 2.5 g / L NaHCO3, 1×GlutaMAX, 10 mM D-glucose, 2% BSA, 0.5×ITS-X, and 100 U / mL penicillin-streptomycin at the final concentration.

[0025] The basal medium used in S4-S5 is: MCDB 131 medium, additionally adding 1.5 g / L NaHCO3, 1×GlutaMAX, 20 mM D-glucose, 2% BSA, 0.5×ITS-X, 10 μM ZnSO4, 10 μg / mL heparin, and 100 U / mL penicillin-streptomycin at the final concentration.

[0026] The second aspect of the present invention provides the application of the above method in inducing islet-like tissues.

[0027] In one embodiment, the application includes the following steps:

[0028] S6 lasted for 3-14 days: Add 10 μM ALK5iII, 2 μM R428, 10 μM vitamin E analogue Trolox, 1 mM N-acetylcysteine, and 1 μM thyroxine T3 at the final concentration to the culture medium. The basal medium used in S6 is: MCDB 131 medium, additionally adding 1.5 g / L NaHCO3, 1×GlutaMAX, 20 mM D-glucose, 2% BSA, 0.5×ITS-X, 10 μM ZnSO4, 10 μg / ml heparin, and 100 U / mL penicillin-streptomycin at the final concentration.

[0029] The third aspect of the present invention provides a method for evaluating the in vitro cell differentiation process, and the method includes the following steps:

[0030] 1) First, in order to balance the number of cells in different cell populations on the path, we perform downsampling on each group of cells, and in principle, make the number of cells close and the network for subsequent analysis stable.

[0031] 2) For the downsampled dataset, calculate the correlation coefficients between all transcription factor genes. Using transcription factor genes as nodes and the values of the correlation coefficients as the weights of the edges, establish a fully connected network;

[0032] 3) Cut off 90 - 95% of the low-weight edges, delete the nodes with too low (<5 - 10%) or too high (>80 - 95%) expression ratios in the cells of the downsampled dataset, sparsify the network, and retain the largest subnetwork after sparsification;

[0033] 4) Use an algorithm to cluster the remaining network, such as the Louvain algorithm, remove the communities related to the cell cycle and batch effects, and merge the remaining communities according to similarity to obtain multiple modules of the network;

[0034] 5) Assess the differentiation efficiency and cell characteristics of the in vitro differentiation process by analyzing whether the three-module co-expression network structure of the in vivo differentiation process is established during the in vitro differentiation process.

[0035] Compared with the prior art, the present invention shows the following remarkable technical progress and beneficial effects:

[0036] 1) By extending the induction duration of the second stage (S2) to 4 - 6 days, this method effectively promotes S2 cells to reach a state similar to AL-P cells in the human body and establishes a stable gene network. Tests find that an induction time of 4 days is optimal for the H1 embryonic stem cell line (H1 ESC).

[0037] 2) On the basis of extending the S2 induction duration, the present invention activates the TGF-β Nodal pathway and the WNT pathway by adding Activin A and CHIR-99021 respectively, strengthening the pancreatic development potential of S2-induced cells and increasing their similarity to AL-P cells in the human body. For H1 ESC, the recommended final concentrations of Activin A and CHIR-99021 are 10 ng / mL and 0.3 μM respectively.

[0038] 3) The present invention replaces the 5-day induction period of S3 and S4 in the control method with the adjusted S3 induction protocol and reduces the FGF10 concentration from 50 ng / mL in the control method to 2 ng / mL. This improvement not only shortens the induction duration and reduces the cost, but also promotes the generation of PP cells more similar to those in vivo, establishes a gene regulatory network closer to in vivo PP cells relative to the control method, and eliminates the potential to differentiate into other endoderm cell lineages.

[0039] 4) The induction process from S1 to S3 not only optimizes the generation of pancreatic progenitor cells (PP), but also directly affects the subsequent differentiation path and efficiency of EP cells. Compared with the control method, a higher proportion of β-like cells and their progenitor cells is obtained, and the generation of multi-hormonal or α-like cells is significantly reduced.

[0040] 5) The islet-like tissue formed by PP cells induced by the present invention exhibits excellent curative effects in a diabetic mouse model, significantly improving the hyperglycemic symptoms.

[0041] 6) Through the detection after transplantation of the islet-like tissues transplanted at different induction stages, it is found that the present invention can reduce the entire induction process to 5 stages in total for 19 days, and the islet-like tissues can still effectively regulate the blood glucose level after transplantation, indicating that the last stage of the control induction method can be omitted in this process.

[0042] 7) Through single-cell transcriptome detection of the grafts, it is found that the present invention can increase the proportion of β cells in the grafts to up to 60%-70%, which is much higher than that in the grafts of the control method, and the cell function is further matured after transplantation.

[0043] 8) The present invention deeply analyzes the path and regulation mechanism of human pancreatic differentiation. Especially by establishing a stable three-module gene co-expression network, it provides a new standard for evaluating the effect of in vitro induction, thereby improving the accuracy and reliability of the method. Description of the Drawings

[0044] The drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0045] Figure 1 Schematic diagrams of the processes of the control method and the present invention.

[0046] Figure 2 Single-cell transcriptome analysis of the pancreatic lineage development during the human embryonic period: (a) Single-cell differentiation path of the human embryonic period from DE cells to each pancreatic lineage. DE, definitive endoderm; MG-P, midgut cells with pancreatic differentiation fate; AL-P, anterior lip cells with pancreatic differentiation fate; DPE, dorsal pancreatic endoderm cells; VPE, ventral pancreatic endoderm cells; PP, pancreatic progenitor cells; Tip, tip cells; Trunk, trunk cells; Acinar, acinar cells; Duct, duct cells; EP, endocrine progenitor cells. SS, somite stage; CS, Carnegie stage; W, weeks post-conception. (b) And Figure 2The corresponding pancreatic lineage differentiation model. (c, d) Transcription factor co-expression network (c) and expression heatmap (d) from DE cells to pancreatic endocrine cells during human embryonic development. I-III represent three gene modules. (e) Relationship tree among MG-P, AL-P, DPE, VPE, and PP-early cells in humans and mice.

[0047] Figure 3 Single-cell transcriptome analysis of the control method: (a) Flow analysis statistics of S1D2 cells. CXCR4, CD117, FOXA2, and SOX17 are characteristic genes of DE. (b) Display of cell types (left) and induction periods (right) in the control method. GI, gastrointestinal cells; EC, enterochromaffin cells. (c) Proportion of various cell types at each induction period in the control method. The colors are consistent with Figure 3 the cell types in b. (d, e) Transcription factor co-expression network (d) and expression heatmap (e) from DE cells to pancreatic endocrine cells in the control method. I-IV represent four gene modules. (f) Similarity analysis of each cell type in the control method with related cell types in vivo. Stomach, gastric cells; Duodenum, duodenal cells; SI, small intestine cells; Liver, liver cells; GI, gastrointestinal cells; EC, enterochromaffin cells.

[0048] Figure 4 Single-cell transcriptome analysis of the method of the present invention: (a) Display of cell types (left) and induction periods (right) in the method of the present invention. (b) Proportion of various cell types at each induction period in the method of the present invention. The colors are consistent with Figure 4 the cell types in a. (c, d) Transcription factor co-expression network (c) and expression heatmap (d) from DE cells to pancreatic endocrine cells in the method of the present invention. I-III represent three gene modules. (e) Similarity analysis of each cell type in the method of the present invention with related cell types in vivo. Stomach, gastric cells; Duodenum, duodenal cells; SI, small intestine cells; Liver, liver cells; GI, gastrointestinal cells; EC, enterochromaffin cells. (f) Comparison of gene network construction of PP cells in vivo, the control method, and the method of the present invention during human embryonic development. (g) Expression of transcription factors from DE to PP cells in the control method and the method of the present invention. Blue words represent the control method, and red words represent the method of the present invention. (h) Comparison of single-cell paths from DE to PP cells in the control method and the method of the present invention. (i) Comparison of single-cell paths from EP to endocrine cells in the control method and the method of the present invention. (j) Expression of transcription factors in EP1-4 cells in the control method and the method of the present invention. Blue words represent the control method, and red words represent the method of the present invention.

[0049] Figure 5Comparison of the effects of different induction durations of S2 in the present invention: (a) Single-cell transcriptome analysis of different induction durations of S2 in the present invention and the control method. (b) Similarity analysis of S2 cells induced for different S2 durations with related cell types in vivo. (c) Gene expression levels in S2 cells induced for different S2 durations. (d) Gene expression levels in S4D3 cells of the control method and S3D3 cells induced by different induction durations of S2 in the present invention.

[0050] Figure 6 Comparison of the effects of adding factors to S2 in the present invention: (a) Single-cell transcriptome analysis of S2D4 cells under different factor treatment conditions. AA represents the addition of Activin A, CH represents the addition of CHIR-99021, and Ctrl represents the control group. (b) Similarity analysis of S2D4 cells (DE4) under different factor treatment conditions with related cell types in vivo. (c) Projection display of DE, AL-L, AL-P, and VPE cells in vivo and S2D4 cells under different factor treatment conditions. (d) Cell ratios under different factor treatment conditions of S2. (e) Gene expression levels in S2D4 cells (DE4) under different factor treatment conditions of S2.

[0051] Figure 7 Functional analysis of islet-like tissues in the control method and the method of the present invention: (a, b) Immunofluorescence staining shows the expression of characteristic proteins in islet-like tissues of the control method and the method of the present invention. (c) Glucose tolerance test analysis of NOD-SCID mice with diabetes models transplanted with S7D14 islet-like tissues of the control method and S6D14 islet-like tissues of the present invention for one month. n represents the number of mice. (d) Fasting blood glucose changes after transplantation of S7D14 islet-like tissues of the control method, S6D14, S6D3, and S5D7 islet-like tissues of the present invention into NOD-SCID mice with diabetes models. Fasting blood glucose is the blood glucose level after 8 hours of starvation. (e) Immunofluorescence staining shows the expression of C-peptide and GCG in the grafts. (f) Smart-seq3 single-cell transcriptome analysis of the grafts one month after islet-like tissue transplantation. UI represents cell types with unclear cell properties. (g) Figure 7 Characteristic gene expression of various cell types in f. (h) Cell ratio composition of the grafts one month after islet-like tissue transplantation. The colors are Figure 7 consistent with the cell types in f. (i) 10x Genomics single-cell transcriptome analysis of S5D7 islet-like tissues of the present invention shows cell types (left) and cell ratios (right). (j) 10x Genomics single-cell transcriptome analysis of the grafts of S5D7 islet-like tissues of the present invention one month after transplantation shows cell types (left) and cell ratios (right). Detailed implementation manners

[0052] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only for illustrating and explaining the present invention, and are not used to limit the present invention.

[0053] The English name abbreviations and their Chinese counterparts used in the embodiments are listed as follows:

[0054]

[0055]

[0056]

[0057]

[0058] Example 1 (comparative example) Existing method for inducing the differentiation of human pluripotent stem cells into pancreatic progenitor cells

[0059] The induction method currently widely used in the field comes from the Timothy Kieffer laboratory, and the optimization schemes of many research groups are also related to this method. These schemes regulate signaling pathways through various combinations of recombinant proteins or small molecules, process for different durations, generally form PE / PP-like cells through four induction stages, and subsequently induce the formation of islet-like tissues or β-like cells through two or three stages. In this process, the cell properties of each differentiation stage in vitro are defined using limited cell type-related characteristic genes.

[0060] The following will describe the 7 stages (Stage, hereinafter referred to as S) of the control method in the present invention. The schematic diagram is shown in Figure 1 a, The whole process is carried out under the culture conditions of 37 °C and 5% carbon dioxide.

[0061] Starting from the human embryonic stem cell line H1, it is maintained in mTeSR1 pluripotent stem cell medium, and TrypLE TM Express enzyme is used to disperse H1 embryonic stem cells into single cells, which are plated onto a pre-prepared culture plate incubated with 1:30 diluted Matrigel. The plating cell concentration is 0.53x10 6 cells / cm 2 , and cultured in mTeSR1 pluripotent stem cell medium supplemented with a final concentration of 10 μM Y-27632. After 24 hours, the following induction differentiation stage is started.

[0062] The basal media for Stage 1 and Stage 2 (S1 and S2) are: MCDB 131 medium supplemented with a final concentration of 1.5 g / L NaHCO3, 1×GlutaMAX, 10 mM D-glucose, 0.5% BSA, 100 U / mL penicillin-streptomycin.

[0063] The basal media for Stages 3 and 4 (S3 and S4) are: MCDB 131 medium supplemented with 2.5 g / L NaHCO3, 1× GlutaMAX, 10 mM D-glucose, 2% BSA, 0.5× ITS-X, and 100 U / mL penicillin-streptomycin.

[0064] The basal media for Stages 5-7 (S5-S7) are: MCDB 131 medium supplemented with 1.5 g / L NaHCO3, 1× GlutaMAX, 20 mM D-glucose, 2% BSA, 0.5× ITS-X, 10 μM ZnSO4, 10 μg / mL heparin, and 100 U / mL penicillin-streptomycin.

[0065] S1 lasts for 2 days (Day, hereinafter referred to as D). By simultaneously activating the TGF-β Nodal pathway (adding 100 ng / mL Activin A at the final concentration) and the WNT pathway (adding CHIR-99021, using 3 μM at the final concentration on D1 and 0.3 μM on D2), it reaches the DE stage of CXCR4+ / FOXA2+ / SOX17+.

[0066] S2 lasts for 2 days. By simultaneously activating the FGF pathway (adding 50 ng / mL FGF10 at the final concentration) and adding 0.25 mM vitamin C at the final concentration, it reaches the PGT stage, and there are no good indicator genes in this stage currently.

[0067] S3 lasts for 2 days. By simultaneously activating the FGF pathway (adding 50 ng / mL FGF10), inhibiting the SHH pathway (adding 0.25 μM SANT-1 at the final concentration), activating the Retinoic acid (RA) pathway (adding 1 μM RA at the final concentration), inhibiting the BMP pathway (adding 100 nM LDN-193189 at the final concentration), activating PKC (adding 200 nM TPPB at the final concentration), and adding 0.25 mM vitamin C at the final concentration, it reaches the PF stage of PDX1+.

[0068] S4 lasts for 3 days. By simultaneously activating the FGF pathway (adding 2 ng / mL FGF10), inhibiting the SHH pathway (adding 0.25 μM SANT-1 at the final concentration), activating the Retinoic acid (RA) pathway (adding 0.1 μM RA at the final concentration), inhibiting the BMP pathway (adding 200 nM LDN-193189 at the final concentration), activating PKC (adding 100 nM TPPB at the final concentration), and adding 0.25 mM vitamin C at the final concentration, it reaches the PE / PP stage of PDX1+ / NKX6-1+.

[0069] At the end of S4, TrypLE TM Express enzyme was used to disperse the cells into single cells, and they were aggregated in 5 mL of S5 medium at a concentration of 5 x 10 6 cells / well on a low-attachment 6-well plate. Y-27632 with a final concentration of 10 μM was added on the first day of S5 to maintain single-cell viability, and the cells were cultured at 100 rpm on an orbital shaker. Then, they were further differentiated into EP cells and islet-like tissue containing β-like cells through three stages.

[0070] S5 lasted for 3 days. The EP stage was achieved by simultaneously inhibiting the SHH pathway (adding a final concentration of 0.25 μM SANT-1), activating the Retinoic acid (RA) pathway (adding a final concentration of 0.05 μM RA), inhibiting the BMP pathway (adding a final concentration of 100 nM LDN-193189), inhibiting the TGF-βRI kinase (adding a final concentration of 10 μM ALK5i II), and adding a final concentration of 1 μM thyroxine T3.

[0071] S6 lasted for 7 days. Immature islet-like tissue was obtained by simultaneously inhibiting the BMP pathway (adding a final concentration of 100 nM LDN-193189), activating the Notch pathway (adding a final concentration of 100 nM GSI XX), inhibiting the TGF-βRI kinase (adding a final concentration of 10 μM ALK5i II), and adding a final concentration of 1 μM thyroxine T3.

[0072] S7 lasted for 14 days. Further mature islet-like tissue was obtained by simultaneously inhibiting the TGF-βRI kinase (adding a final concentration of 10 μM ALK5i II), inhibiting the tyrosine protein kinase receptor (adding a final concentration of 2 μM R428), and adding a final concentration of 10 μM vitamin E analogue Trolox, 1 mM N-acetylcysteine, and 1 μM thyroxine T3.

[0073] Example 2 Improved method for inducing the differentiation of human pluripotent stem cells into pancreatic progenitor cells

[0074] The present invention also used the human embryonic stem cell line H1 as the starting cell for culture, maintained the culture in mTeSR1 pluripotent stem cell medium, and used TrypLE TM Express enzyme to disperse the H1 embryonic stem cells into single cells, which were plated onto a pre-prepared culture plate incubated with 1:30 diluted Matrigel, and the plated cell concentration was 0.53 x 10 6 cells / cm 2, cultured in mTeSR1 pluripotent stem cell medium supplemented with a final concentration of 10 μM Y-27632, and induction of differentiation was initiated 24 hours later. The induction of human pluripotent stem cells into pancreatic progenitor cells (PP) in the present invention is carried out in a total of 3 stages, and on this basis, 3 more induction stages are continued to obtain islet-like tissue. The whole process is carried out under the culture conditions of 37 °C and 5% carbon dioxide. The schematic diagram is shown in Figure 1 b.

[0075] The basal media for S1 and S2 are the same as those for S1 and S2 in the control method, which are: MCDB 131 medium supplemented with a final concentration of 1.5 g / L NaHCO3, 1× GlutaMAX, 10 mM D-glucose, 0.5% BSA, 100 U / mL penicillin-streptomycin.

[0076] The basal media for S3 are the same as those for S3 and S4 in the control method, which are: MCDB 131 medium supplemented with a final concentration of 2.5 g / L NaHCO3, 1× GlutaMAX, 10 mM D-glucose, 2% BSA, 0.5× ITS-X, 100 U / mL penicillin-streptomycin.

[0077] The basal media for S4 - S6 are the same as those for S5 - S7 in the control method, which are: MCDB 131 medium supplemented with a final concentration of 1.5 g / L NaHCO3, 1× GlutaMAX, 20 mM D-glucose, 2% BSA, 0.5× ITS-X, 10 μM ZnSO4, 10 μg / mL heparin, 100 U / mL penicillin-streptomycin.

[0078] S1 lasts for 2 days, by simultaneously activating the TGF-β Nodal pathway (adding a final concentration of 100 ng / mL Activin A) and activating the WNT pathway (adding CHIR-99021, with a final concentration of 3 μM used on D1 and 0.3 μM used on D2).

[0079] S2 lasts for 4 days, by simultaneously activating the FGF pathway (adding a final concentration of 50 ng / mL FGF10), activating the TGF-β Nodal pathway (adding a final concentration of 10 ng / mL Activin A), activating the WNT pathway (adding a final concentration of 0.3 μM CHIR-99021), and adding a final concentration of 0.25 mM vitamin C.

[0080] S3 lasted for 3 days, by simultaneously activating the FGF pathway (adding FGF10 at a final concentration of 2 ng / mL), inhibiting the SHH pathway (adding SANT-1 at a final concentration of 0.25 μM), activating the Retinoic acid (RA) pathway (adding RA at a final concentration of 1 μM), inhibiting the BMP pathway (adding LDN-193189 at a final concentration of 100 nM), activating PKC (adding TPPB at a final concentration of 200 nM) and adding vitamin C at a final concentration of 0.25 mM.

[0081] At the end of S3, TrypLE TM Express enzyme was used to disperse the cells into single cells, and they were aggregated in 5 mL of S4 medium at a concentration of 5x10 6 cells / well on a low-attachment 6-well plate. Y-27632 at a final concentration of 10 μM was added on the first day of S4 to maintain single-cell viability, and the cells were cultured at 100 rpm on an orbital shaker.

[0082] The induction protocols for S4-S6 were the same as those for S5-S7 of the control method, but the duration of S6 in the present invention could be shortened or omitted.

[0083] S4 lasted for 3 days, by simultaneously inhibiting the SHH pathway (adding SANT-1 at a final concentration of 0.25 μM), activating the Retinoic acid (RA) pathway (adding RA at a final concentration of 0.05 μM), inhibiting the BMP pathway (adding LDN-193189 at a final concentration of 100 nM), inhibiting the TGF-βRI kinase (adding ALK5i II at a final concentration of 10 μM) and adding thyroxine T3 at a final concentration of 1 μM.

[0084] S5 lasted for 7 days, by simultaneously inhibiting the BMP pathway (adding LDN-193189 at a final concentration of 100 nM), activating the Notch pathway (adding GSI XX at a final concentration of 100 nM), inhibiting the TGF-βRI kinase (adding ALK5i II at a final concentration of 10 μM) and adding thyroxine T3 at a final concentration of 1 μM.

[0085] S6 lasted for 3 - 14 days, by simultaneously inhibiting the TGF-βRI kinase (adding ALK5i II at a final concentration of 10 μM), inhibiting the tyrosine protein kinase receptor (adding R428 at a final concentration of 2 μM), and adding the vitamin E analogue Trolox at a final concentration of 10 μM, 1 mM N-acetylcysteine, and thyroxine T3 at a final concentration of 1 μM.

[0086] Results analysis of Example 3

[0087] 1. Evaluate the induction effects of the two methods using the in vivo pancreatic differentiation pathway and gene network respectively:

[0088] By analyzing the single-cell transcriptome of the pancreas during the embryonic period (from 2 somites to 19 weeks of gestation) in humans, the cell types, differentiation pathways, and gene networks of the pancreatic lineage were resolved ( Figure 2 a-d), which were used as criteria for evaluating the in vitro induction effect. Analysis revealed that during the development from DE cells to endocrine cells in the human body, the gene network could be divided into 3 modules, and PP cells were in a crucial position connecting the transition between the first and second modules ( Figure 2 c, d). Moreover, in vivo, PP cells were developed from the ventral (DE–AL-P–VPE–PP) and dorsal (DE–MG-P–DPE–PP) endoderm pathways respectively and converged ( Figure 2 a, b), and the cells on the ventral pathway (AL-P and VPE) were more similar to PP cells than the cells on the dorsal pathway (MG-P and DPE), which was not the case in mice ( Figure 2 e). This suggests that mimicking the ventral developmental pathway in the human body is a more efficient way to form PP cells.

[0089] (1) Starting from the human embryonic stem cell line H1, directed differentiation was carried out using the control method in Example 1. Flow cytometry analysis showed that at the end of S1D2, the proportions of CXCR4+CD117+ cells and FOXA2+SOX17+ cells could reach more than 97% ( Figure 3 a). At the same time, Smart-seq3 single-cell transcriptome analysis was performed on the differentiated cells. Analysis found that the control method could be divided into 20 cell types throughout the differentiation process ( Figure 3 b). Among them, the β-like cells (SC-β) identified in the final stage S7 account for 18% - 29%, the α-like cells (SC-α) account for 37% - 64%, the δ-like cells (SC-δ) account for 2% - 5%, EC1 accounts for 5% - 15%, and EC2 accounts for 1% - 5%, EC3 (β progenitor cells) accounts for 0.8% - 2% ( Figure 3 c), SC-β and its progenitor cells together account for 19% - 32% . From the perspective of the gene co-expression network, during the in vitro differentiation from DE cells to endocrine cells, the gene network could be divided into 4 modules ( Figure 3 d, e), which indicates the differences in gene network construction between the control scheme and the in vivo development process. From the similarity between in vivo and in vitro cells, the PE and PE / PP cells obtained by the control method had properties similar to both in vivo DPE and VPE, and the obtained PP cells, in addition to having a certain similarity to in vivo PP cells, also had a strong similarity to gastrointestinal cells ( Figure 3 f), which indicates that the cell properties induced by the control scheme were relatively mixed, and the unstable construction of the gene network led to unstable cell states. In addition, EC3 cells had similarities to in vivo β cells, and combining the characteristic gene expression and path characteristics, they were considered to be β progenitor cells, while EC1 / 2 had similarities to in vivo EC cells.

[0090] (2) Through the analysis of Example 1, it was found that there was a problem with the establishment of the gene network during the current induction process. Starting from the human embryonic stem cell line H1, the method of the present invention (Example 2) was used for directed differentiation, and Smart-seq3 single-cell transcriptome analysis was performed on the differentiated cells. The analysis found that the method of the present invention could be divided into 20 cell types during the entire differentiation process ( Figure 4 a). Among them, the β-like cells (SC-β) identified in the final stage S6 account for 55% - 68%, and the α-like cells (SC-α) account for 11% - 21%, the δ-like cells (SC-δ) account for 0.8% - 2%, EC1 accounts for 11% - 17%, EC2 accounts for 0% - 3%, and EC3 (β precursor cells) Accounting for 2%-5% ( Figure 4 b), SC-β and its precursor cells together account for 60%-70% . Compared with the control method, the cell proportions of SC-β and EC3 (β precursor cells) were significantly increased, the cell proportion of SC-α was significantly decreased, and the cell proportion of SC-δ was slightly decreased. From the perspective of the gene co-expression network, during the differentiation process from DE cells to endocrine cells in vitro by the method of the present invention, the gene network could be divided into 3 modules ( Figure 4 c, d), which was similar to the transformation during the in vivo development process. From the similarity between in vivo and in vitro cells, the DE4 cells obtained by the method of the present invention at S2D4 were similar to AL-P in vivo, the obtained PP cells were similar to the PP cells in vivo, and did not have similarity with the gastrointestinal cells in vivo ( Figure 4 e), indicating that the gene network established by the induction of the present invention was stable, and the cell properties were clear and stable. In addition, by comparing the gene network formed with the PP cells in vivo, the method of the present invention could better construct the gene network connection of the in vivo PP cells compared with the control method, and had higher expression of the characteristic genes related to PP cells, such as SOX9, PDX1, NKX6-1, and PTF1A, etc. ( Figure 4 f, g). By comparing the differentiation paths of the control method and the method of the present invention, it was found that starting from the optimized treatment of S2, the two methods showed different differentiation paths, including the path of PP generation and the path of EP differentiation ( Figure 4 h, i). In addition, the EP cells generated by the method of the present invention highly expressed the regulatory transcription factors related to β cells, such as PAX4, PDX1, NKX6-1, and MAFB, while the EP cells in the control method highly expressed the regulatory transcription factors related to α cells, such as ARX and ETV1, indicating that the EP cells of the two methods had different endocrine lineage differentiation tendencies ( Figure 4 j).

[0091] 2. Prolonging the induction duration of S2 can enhance the similarity between induced DE cells and in vivo AL-P cells

[0092] Starting from the human embryonic stem cell line H1, the conditions for S2 were as follows: MCDB 131 medium supplemented with 1.5 g / L NaHCO3, 1× GlutaMAX, D-glucose at a final concentration of 10 mM, 0.5% BSA, 100 U / mL penicillin-streptomycin, 50 ng / mL FGF10, 10 ng / mL Activin A, 0.3 μM CHIR-99021, and 0.25 mM vitamin C. S2 was carried out for 3, 4, 5, and 6 days respectively (denoted as groups A - D), and then continued to be directed differentiated to S3 according to the induction method of S3 in Example 2. Smart-seq3 single-cell transcriptome analysis was performed on the cells at the end of S2 and S3 respectively ( Figure 5 a). At the same time, directed differentiation to S4 was carried out using the control method of Example 1, and Smart-seq3 single-cell transcriptome analysis was performed on the cells at the end of each stage from S2 to S4 ( Figure 5 a).

[0093] Analysis found that compared with the cells treated with S2 for 3 days, the cells treated with S2 for 4 - 6 days had stronger similarity to in vivo AL-P cells ( Figure 5 b). Among them, 4 days of S2 induction was the optimal duration. S2D4 up-regulated the expression of transcription factors SOX4, HHEX, PROX1, and ONECUT1 related to AL-P, and down-regulated the expression of transcription factors SOX17, GATA4, and PITX2 related to DE and MG-P cells ( Figure 5 c). Continuing to extend S2 to 5 days and 6 days could not further enhance the properties of AL-P, and would lead to the down-regulation of the PP cell-related transcription factor PDX1 and the up-regulation of the non-PP cell-related gene AFP at S3D3 ( Figure 5 d).

[0094] 3. S2 activates the TGF-β Nodal pathway and the WNT pathway to enhance the similarity between induced DE cells and in vivo AL-P cells

[0095] Starting from the human embryonic stem cell line H1, S2 lasted for 4 days under the conditions of: MCDB 131 medium supplemented with 1.5 g / L NaHCO3, 1× GlutaMAX, D-glucose at a final concentration of 10 mM, 0.5% BSA, 100 U / mL penicillin-streptomycin, 50 ng / mL FGF10 and 0.25 mM vitamin C (for the Ctrl group), or different concentrations of TGF-β Nodal pathway activator (final concentration of 10 ng / mL, 25 ng / mL, 50 ng / mL Activin A, denoted as AA1 - AA3 groups respectively) or WNT pathway activator (final concentration of 0.3 μM, 1 μM, 2 μM CHIR-99021, denoted as CH1 - CH3 groups respectively) were added simultaneously, and Smart-seq3 single-cell transcriptome analysis was performed on the cells at S2D4 ( Figure 6 a).

[0096] Analysis found that adding CHIR-99021 could promote the differentiation of DE4 cells at S2D4 into AL-P and AL cells (AL-L) in the in vivo liver direction, while adding Activin A could effectively inhibit the differentiation in the AL-L direction ( Figure 6 b, c). With the increase in the concentration of CHIR-99021 added, the proportion of off-target cells would increase. In addition, adding Activin A would also delay the shutdown of the expression of related genes in DE cells and slow down the differentiation process ( Figure 6 b - d). Therefore, using the combination of the optimal concentrations of these two factors, 10 ng / mL Activin A and 0.3 μM CHIR-99021, and their functional hedging, the early cells were inhibited from differentiating in the liver direction and promoted to differentiate in the AL-P cell direction, inhibiting the expression of liver differentiation-related genes such as AFP, FGB, and AMBP, as well as DE-related genes PITX2 and GATA4 ( Figure 6 e).

[0097] 4. Functional detection of induced islet-like tissue in the present invention (Example 2) and control method (Example 1)

[0098] Starting from the human embryonic stem cell line H1, directional differentiation was carried out using the present invention (Example 2) and the control method (Example 1). Immunofluorescence staining detection was performed on the islet-like tissue at the final 14-day induction stage, and it was found that the islet-like tissue of the present invention had a high proportion of PDX1+NKX6-1+C-peptide+ cells, and the proportion of GCG+ cells decreased ( Figure 7 a, b), which was consistent with the analysis results of the single-cell transcriptome data.

[0099] In addition, islet-like tissues that each induced the final stage (S7D14 of the control method, S6D14 of the present invention) were transplanted into immunodeficient mice NOD-SCID with diabetes model, and about 2.5 million cells were transplanted into each mouse. NOD-SCID mice were purchased from Vital River and a diabetes mouse model was constructed by injecting low-dose (35 mg / kg body weight) streptozotocin (STZ) continuously for 5 days. Experimental findings showed that the islet-like tissues obtained by the method of the present invention had better blood glucose lowering effects and faster blood glucose lowering functions in the glucose tolerance test one month after transplantation ( Figure 7 c, d). By recovering the grafts one month after transplantation and detecting them by immunofluorescence staining, it was found that the grafts of the method of the present invention had a higher proportion of C-peptide+ cells, and a high proportion of such cells could be stably observed in the grafts six months after transplantation ( Figure 7 e). By performing Smart-seq3 single-cell transcriptome analysis on the grafts one month after transplantation, it was found that there were various cell types in the grafts, including SC-β, SC-α, SC-δ and EC cells, as well as a group of cell types with unclear cell properties (UI, unidentified), which co-expressed PDX1 and exocrine characteristic genes such as CPA2 and SOX9 ( Figure 7 f, g). Among them, In the graft of S6D14 of the present invention, SC-β, SC-α, SC-δ and EC account for 48.8%, 29.0%, 8.8% and 12.5% respectively, and less than 1% of UI cells; while in the graft of the control method S7D14 SC-β, SC-α, SC-δ and EC account for 8.9%, 70.0%, 1.8% and 3.4% respectively, and up to 15.9% of UI cells cells ( Figure 7 h). The grafts of the present invention had a higher proportion of β-like cells with C-peptide+ and a lower proportion of α-like cells with GCG+, but there were no obvious differences in cell properties at the transcriptome level compared with the control method.

[0100] In view of the fact that the proportion of SC-β cells was already very high at S5D7 of the method of the present invention, islet-like tissues at the early stage (S5D7 and S6D3) of the method of the present invention were further transplanted into immunodeficient mice NOD-SCID with diabetes model (the same as the above method). Functional detection found that mice transplanted with S5D7 and S6D3 islet-like tissues had better blood glucose regulation effects compared with mice transplanted with S6D14 islet-like tissues ( Figure 7 c, d), and Smart-seq3 single-cell transcriptome analysis also showed that there was a higher proportion of SC-β cells (67%) in its grafts ( Figure 7 h). In addition, the analysis found that although there were some differences in the transcriptome between the cells at S5D7 and the cells at S6 before transplantation of the method of the present invention, there were no obvious differences after transplantation ( Figure 3a, 7f), indicating that after transplantation into the body, it can significantly promote the maturation of islet-like tissues, eliminating the relatively immature differences during in vitro induction. By performing 10x Genomics single-cell transcriptome analysis on the S5D7 islet-like tissues before transplantation of the method of the present invention and their grafts one month after transplantation, it was found that the cell types and proportions were consistent with those of Smart-seq3 single-cell transcriptome analysis ( Figure 7 i, j). By performing Smart-seq3 single-cell transcriptome analysis on the grafts after transplantation of the early S6D7 islet-like tissues by the control method, it was found that the UI cells therein were as high as 46% ( Figure 7 h), indicating that reducing one period by the control method would lead to incomplete differentiation of the endocrine lineage and could not achieve the differentiation effect after shortening one period as in the present invention. In summary, the method of the present invention can significantly shorten the induction duration to as few as 5 induction stages for a total of 19 days, obtaining islet-like tissues with better function after transplantation.

[0101] Example 4 Analysis of gene co-expression network

[0102] During development, the rate of cell fate transition is not constant, but there are alternations between smooth transitions and jumps. Among them, the transition of gene modules in the transcription factor co-expression network represents the jump of cell fate, and the number of modules reflects the number of stages of smooth transition of cell states during development. The present invention deeply analyzed the dynamic changes of the transcription factor co-expression network during the development from DE to the endocrine lineage in the human embryo through single-cell transcriptome analysis, revealing its three-module structure ( Figure 2 c, d). The first module covers the differentiation from DE to PP-early cells, and the cells gradually turn off the gene expression related to the endoderm and turn on the gene expression related to pancreatic differentiation. The jump between the first and second modules indicates that the loss of co-expression characteristics of transcription factors related to pancreatic differentiation and transcription factors related to endoderm cell identity in the first module, establishing the co-expression characteristics of transcription factors related to the identity of endocrine progenitor cells, and smoothly transitioning to endocrine progenitor cells. The transition between the second and third modules represents the regulatory transition of the endocrine lineage fate.

[0103] The analysis of the transcription factor co-expression network in the in vivo process of the present invention was completed by the following bioinformatics analysis methods:

[0104] 1) First, in order to balance the number of cells in different cell populations on the path, we Figure 2 randomly sampled each of the 21 cell populations identified in a to achieve downsampling. If the number of cells in a cell population is greater than 80, then 80 cells are randomly selected from it; if it is less than or equal to 80, then all cells are retained.

[0105] 2) For the downsampled dataset, calculate the Pearson correlation coefficients between all transcription factor genes using R software. Taking transcription factor genes as nodes and the values of the correlation coefficients as the weights of the edges, establish a fully connected network.

[0106] 3) Cut off the edges with weights less than 0.25, delete the nodes with expression proportions lower than 10% or higher than 80% in the cells of the downsampled dataset, and then remove the unconnected nodes and sub-networks with less than 10 genes.

[0107] 4) Use the Louvain algorithm in igraph software to cluster the remaining network, remove the communities related to the cell cycle and batch effects, and merge the remaining communities according to similarity to obtain multiple modules of the network. Considering the inherent differences in gene expression between the in vivo development system and the in vitro directed induction system, the inventors believe that using the module architecture of the in vivo gene co-expression network as the standard for comparison with the in vitro system is more reliable and accurate, and can be used to evaluate the in vitro differentiation process from a global perspective of cell fate regulation transformation.

[0108] By performing a similar analysis as above on the in vitro differentiation process from embryonic stem cells to endocrine cells, the inventors found that the control method presented the characteristics of a four-module gene co-expression network ( Figure 3 d, e), which did not match the in vivo characteristics, and its unstable early gene network was exactly the core to be overcome by the present invention. Through analysis, it was found that the method of the present invention presented a three-module gene co-expression network consistent with the in vivo ( Figure 4 c, d).

[0109] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and its equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A method for evaluating an in vitro cell differentiation process, characterized in that, The method includes the following steps: 1) First, in order to balance the number of cells in different cell populations on the path, we perform downsampling on each cell population to make the number of cells close and the network for subsequent analysis stable; 2) For the downsampled dataset, calculate the correlation coefficients between all transcription factor genes, use the transcription factor genes as nodes, and the values of the correlation coefficients as the weights of the edges to establish a fully connected network; 3) Cut off 90-95% of the low-weight edges, delete the nodes with an expression ratio lower than 10% or higher than 80% in the cells of the downsampled dataset to sparsify the network, and retain the largest subnetwork after sparsification; 4) Use an algorithm to cluster the remaining network, remove the communities related to the cell cycle and batch effects, and merge the remaining communities according to similarity to obtain multiple modules of the network; 5) Evaluate the differentiation efficiency and cell characteristics of the in vitro differentiation process by analyzing whether the three-module co-expression network structure of the in vivo differentiation process is established during the in vitro differentiation process.

2. The method according to claim 1, characterized in that, In step 3), the deletion of the nodes with an expression ratio lower than 10% or higher than 80% in the cells of the downsampled dataset refers to the nodes with an expression ratio of 5-10% or 80-95%.

3. The method according to claim 1, characterized in that, The algorithm described in step 4) includes the Louvain algorithm.

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