An induction medium and an induction method for directional differentiation of human amniotic mesenchymal stem cells into liver organoids

Abstract

This method discloses a technique for inducing the directed differentiation of human amniotic mesenchymal stem cells into liver organoids, comprising the following steps: S1, expanding and culturing third-generation human amniotic mesenchymal stem cells; S2, discarding the culture supernatant from step S1 and adding a first induction medium; S3, discarding the culture supernatant from step S2 and adding a second induction medium; S4, discarding the culture supernatant from step S3, adding trypsin to digest the cells, resuspending the cells in a third induction medium, centrifuging, and culturing; S5, after the completion of step S4, discarding the culture supernatant and adding a fourth induction medium, ultimately obtaining functionally mature liver organoids. This induction method avoids the tumorigenicity problems associated with other methods, such as induced pluripotent stem cell induction, making it more suitable for industrialization and clinical application. Furthermore, it uses simple components and has lower technical requirements, thus greatly increasing the feasibility of the experiment and facilitating industrialization.

Description

Technical Field

[0001] This invention relates to the field of regenerative medicine technology, specifically to an induction culture medium and induction method for the directed differentiation of human amniotic mesenchymal stem cells into liver organoids. Background Technology

[0002] Regenerative medicine is a discipline dedicated to repairing, replacing, or regenerating damaged tissues and organs. Stem cell technology, as its core support, provides a new direction for the treatment of end-stage organ diseases. Mesenchymal stem cells (MSCs) are a type of adult mesenchymal cell with self-renewal capacity and multi-lineage differentiation potential. Due to their wide availability, low immunogenicity, and minimal ethical controversy, they have become a research hotspot in the field of regenerative medicine.

[0003] Human amniotic mesenchymal stem cells (hAMSCs) are a branch of mesenchymal stem cells and belong to the category of adult stem cells. Human amniotic membrane is a highly abundant and readily available tissue. Since it is discarded after childbirth, there are no ethical concerns, making it suitable for regenerative medicine and cell therapy. Typically, after delivery, the placenta is discarded, the amniotic membrane tissue is bluntly dissected, and then digested using trypsin and collagenase at specific concentrations, followed by filtration through a steel mesh to obtain hAMSCs. This non-invasive collection method avoids ethical issues. hAMSCs express embryonic stem cell surface markers (Oct-3 / 4, SSEA-3, SSEA-4, Rex-1), with Oct-4 considered a gene for pluripotent stem cell self-renewal and multi-lineage differentiation potential. Due to their multi-lineage differentiation potential, hAMSCs are commonly identified through directed induction and detection, typically involving osteogenic, adipogenic, and chondrogenic induction. Compared to MSCs from other sources such as bone marrow or adipose tissue, human amniotic mesenchymal stem cells (hAMSCs) exhibit higher proliferation rates, lower risk of immune rejection, and greater availability. Under appropriate in vitro conditions, hAMSCs have the potential to differentiate into different tissue cell types from the three germ layers, such as: nerve cells (ectoderm); osteoblasts, chondrocytes, adipocytes, vascular endothelial cells, and cardiomyocytes (mesoderm); and hepatoid cells and pancreatic islet cells (endoderm). hAMSCs do not express major histocompatibility class II antigens (HLA-DP, HLA-DQ, HLA-DR), but express trace amounts of major histocompatibility class I antigens (HLA-A, HLA-B, HLA-C), and also do not express co-stimulatory molecules (CD80, CD83, CD86, CD40L, etc.), indicating that hAMSCs have low immunogenicity. In addition to the above advantages, hAMSCs also have the advantages of being easy to extract and culture, having no ethical restrictions, paracrine properties, promoting angiogenesis, and reducing immune rejection. In conclusion, hAMSCs are an important source of stem cells in the field of regenerative medicine. Due to their convenient acquisition methods and multi-lineage differentiation potential, they have great potential in basic research and clinical applications of regenerative medicine.

[0004] The liver is a vital metabolic and detoxification organ in the human body. End-stage treatments for various liver diseases are very limited, with liver transplantation currently being the only effective radical cure. However, it faces challenges such as donor shortages, immune rejection, and high surgical risks. Liver organoids, as three-dimensional tissue models cultured in vitro, possess structures and functions similar to the in vivo liver. They can be used not only for studying the pathogenesis of liver diseases, drug screening, and toxicity evaluation, but also as carriers for cell transplantation, providing new strategies for the treatment of liver diseases. Therefore, how to efficiently induce stem cells to differentiate into liver organoids has become a key research direction in the field of regenerative medicine.

[0005] The definitive endoderm (DE) is an important stage in embryonic development. DE cells can further differentiate into cells of tissues such as the liver, pancreas, lungs, and intestines. Currently, the identification of DE cells is mainly achieved by detecting the sex-determining region Y-box 17 (SOX17) and the forkhead box A2 (FOXA2).

[0006] The development of the hepatic endoderm (HE) is a complex process, formed from the mature endoderm under the regulation of multiple signaling pathways and transcription factors. Early studies confirmed that fibroblast growth factors (FGFs) play a crucial role in the initiation of differentiation of mammalian endoderm into liver tissue. The action of BMP signaling is parallel to the FGF signaling pathway derived from the cardiogenic mesoderm, both inducing the expression of liver-specific genes in endoderm cells and inhibiting their differentiation into the pancreatic lineage. HE cell identification mainly utilizes HE cell markers such as alpha-fetoprotein (AFP), albumin (ALB), hepatocyte nuclear factor 4α (HNF-4α), and cytokeratin 19 (CK19).

[0007] According to existing literature, induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) can be induced to differentiate into degenerative (DE) cells and hepatocellular carcinoma (HE) cells in vitro. However, both types of cells have certain limitations: iPSCs, due to their unlimited proliferative potential, exhibit severe tumorigenicity, especially during cell transplantation, where even a small amount of residual iPSCs can lead to teratoma formation. Furthermore, iPSCs also present issues such as immunogenicity and cellular heterogeneity; while the acquisition of ESCs raises ethical concerns, thus limiting the widespread application of both cell types. Some literature indicates that mesenchymal stem cells derived from adipose tissue, bone marrow, dental pulp, and umbilical cord can be induced to differentiate into DE and HE cells, ultimately forming liver organoids, but these methods suffer from low differentiation efficiency and incomplete differentiation. Summary of the Invention

[0008] The present invention aims to provide an induction culture medium and induction method for the directed differentiation of human amniotic mesenchymal stem cells into liver organoids. By combining different induction factors and optimizing culture conditions, the directed differentiation from stem cells into mature liver organoids can be achieved.

[0009] To achieve the above objectives, this application provides the following technical solution: A first aspect of the present invention provides an induction culture medium for the directed differentiation of human amniotic mesenchymal stem cells into liver organoids, comprising, according to the cell differentiation stage: First induction medium: composed of fetal bovine serum, activin A, B27 and RPMI 1640 medium; The second induction medium was a mixture of BMP4, bFGF, B27 and RPMI 1640 medium. The third induction medium is a mixture of HGF, oncostatin-M, B27, dexamethasone, and RPMI 1640 medium. The fourth induction medium is a mixture of HGF, oncostatin-M, B27, dexamethasone, and HCM medium.

[0010] A second aspect of the present invention provides a method for culturing a medium obtained from the induction medium described in the first aspect, comprising the following steps: Preferably, in the first induction medium, the final concentration of fetal bovine serum is 0.3%–0.7%, the final concentration of activin A is 90–110 ng / mL, and the final concentration of B27 is 1×; in the second induction medium, the final concentration of BMP4 is 15–25 ng / mL, the final concentration of bFGF is 90–110 ng / mL, and the final concentration of B27 is 1×; in the third induction medium, the final concentration of HGF is 8–12 ng / mL, the final concentration of oncostatin-M is 18–22 ng / mL, the final concentration of B27 is 1×, and the final concentration of dexamethasone is 90–110 nM; in the fourth induction medium, the final concentration of HGF is 8–12 ng / mL, the final concentration of oncostatin-M is 18–22 ng / mL, the final concentration of B27 is 1×, and the final concentration of dexamethasone is 90–110 nM.

[0011] In some embodiments, the final concentrations of fetal bovine serum in the first induction medium are 0.5%, activin A is 100 ng / mL, and B27 is 1×; the final concentrations of BMP4, bFGF, and B27 are 20 ng / mL, 100 ng / mL, and 1× in the second induction medium; the final concentrations of HGF, oncostatin-M, B27, and dexamethasone are 100 nM in the third induction medium; and the final concentrations of HGF, oncostatin-M, B27, and dexamethasone are 100 nM in the fourth induction medium.

[0012] Preferably, the culture medium replacement time for each step is as follows: during the culture period of step S2, the first induction medium is replaced with fresh medium once; during the culture period of step S3, the second induction medium is replaced with fresh medium once; during the culture period of step S4, the third induction medium is replaced with fresh medium every 2 days; and during the culture period of step S5, the fourth induction medium is replaced with fresh medium every 2 days.

[0013] Working principle and beneficial effects of the present invention: This application selects human amniotic mesenchymal stem cells as the starting cells. These cells are adult stem cells and do not have the potential for unlimited proliferation. There is no risk of tumorigenesis during the induction process, thus avoiding the defects of the iPSCs induction system. At the same time, the cells are derived from postpartum placental waste, which avoids ethical controversies and solves the application limitations of ESCs. The phased induction system follows the physiological laws of embryonic liver development. By precisely controlling inducing factors and culture conditions, it achieves directed differentiation from human amniotic mesenchymal stem cells to shaped endoderm, hepatic endoderm, and mature liver organoids. The induced shaped endoderm cells highly express SOX17 and FOXA2 proteins, while the hepatic endoderm cells highly express markers such as AFP and ALB. The resulting liver organoids can stably secrete ALB protein and have high functional maturity. Human amniotic mesenchymal stem cells themselves have low immunogenicity, and the induced liver organoids inherit this advantage. Cell transplantation is less likely to trigger an immune rejection reaction, providing a safe and effective cell carrier for cell therapy of liver diseases, with broad clinical application prospects. Moreover, the components used are simple and the technical requirements are low, which greatly increases the feasibility of the experiment and makes it easy to industrialize. Attached Figure Description

[0014] Figure 1 This is a morphological image of the selected third-generation hAMSCs. Figure 2This is a morphological diagram of DE cells induced from the differentiation of third-generation hAMSCs cells. Figure 3 This is a comparison of the relative expression levels of SOX17 and FOXA2 proteins obtained from hAMSCs cells and DE cells; Figure 4 This is a statistical comparison of the relative expression levels of SOX17 and FOXA2 proteins obtained from hAMSCs cells and DE cells; Figure 5 This is a comparison of the SOX17 and FOXA2 immunofluorescence results obtained from hAMSCs cells and DE cells; Figure 6 This is a statistical comparison of the fluorescence intensity of SOX17 and FOXA2 obtained from hAMSCs cells and DE cells; Figure 7 This is a morphological diagram of HE cells formed through the continued induction and differentiation of DE cells. Figure 8 This is a comparison of E-cadherin fluorescence results obtained from DE cells and HE cells; Figure 9 It represents the ALB gene mRNA transcription level obtained from DE cells and HE cells; Figure 10 It represents the HNF-4α gene mRNA transcription level obtained from DE cells and HE cells; Figure 11 It represents the AFP gene mRNA transcription level obtained from DE cells and HE cells; Figure 12 This refers to the CK19 gene mRNA transcription level obtained from DE cells and HE cells; Figure 13 This is a diagram of organoid morphology formed through continued induction and differentiation of HE cells; Figure 14 This is a statistical graph of ALB protein secretion obtained from liver organoid culture supernatant by ELISA detection. Detailed Implementation

[0015] The following detailed description illustrates the specific implementation method: 1. A method for inducing the directed differentiation of human amniotic mesenchymal stem cells into a defined endoderm, comprising the following steps: S1: (as shown) Figure 1 (As shown) Third-generation human amniotic mesenchymal stem cells were seeded and cultured in 24-well plates, achieving a growth density of 80%. S2: When the human amniotic mesenchymal stem cell growth density reaches 80% as described in step S1, aspirate the supernatant from the culture medium in the 24-well plate, wash three times with PBS buffer, then aspirate the PBS buffer completely. Add fetal bovine serum, activin A, and B27 to RPMI 1640 medium, gently pipette to mix, and obtain the first induction medium. The final concentrations of fetal bovine serum, activin A, and B27 in the first induction medium are 0.5%, 100 ng / ml, and 1×, respectively. Add the obtained first induction medium to the 24-well plate and incubate at 37°C in a 5% CO2 incubator for 6 days, changing the medium once on the 3rd day. Under a light microscope, the cells gradually become flattened, appearing plate-like or fish-scale-like (e.g., ...). Figure 2 (As shown).

[0016] Western blotting and immunofluorescence assays were performed on SOX17 and FOXA2 proteins. The results (i.e., post-induction results) are as follows: Figure 3-6 As shown, surface DE cells highly express the proteins SOX17 and FOXA2, indicating that they differentiate from human amniotic mesenchymal stem cells into the morphological endoderm.

[0017] 2. A method for inducing the directional differentiation of a fixed-type endoderm into hepatic endoderm, comprising the following steps: After 6 days of cell culture as described in step S2, the supernatant from the 24-well plate was aspirated, washed three times with PBS buffer, and then the PBS buffer was completely aspirated. BMP4, bFGF, and B27 were added to RPMI 1640 medium and gently mixed to obtain the second induction medium. The final concentrations of BMP4 and bFGF in the second induction medium were 20 ng / ml, 100 ng / ml, and 1%, respectively. The cells were then placed in a 2.5L anaerobic culture bag, along with a 2.5L microaerophilic gas generator, and cultured at 37°C in a 5% CO2 incubator for 6 days. The medium and gas generator were replaced on the third day. After induction, DE cells were observed under a light microscope to be in a single-layer or multi-layered polygonal shape (e.g., ...). Figure 7 (As shown).

[0018] Real-time quantitative PCR was performed to detect AFP, ALB, HNF-4α, and CK19, as well as E-cadherin immunofluorescence assay. Results (i.e., post-induction results) are as follows: Figure 8-12 As shown, the surface of HE highly expresses the fluorescent signal E-cadherin and HE highly expresses the markers ALB, HNF-α, AFP and CK19, indicating that it differentiates from the fixed endoderm into the liver endoderm.

[0019] 3. A method for inducing the directed differentiation of liver endoderm into liver organoids and their maturation, comprising the following steps: S4: After 6 days of cell culture as described in step S3, aspirate the supernatant from the 24-well plate, wash three times with PBS buffer, then aspirate the PBS buffer completely, treat with trypsin at 37°C for 3–4 minutes, centrifuge at 1000 rpm for 3 minutes, add HGF, oncostatin-M, B27, and dexamethasone to RPMI 1640 medium, gently pipette to mix, and obtain the third induction medium. The final concentrations of HGF, oncostatin-M, and dexamethasone in the third induction medium are 10 ng / ml, 20 ng / ml, 1%, and 100 nM, respectively. Seed the dissociated cells at a ratio of 2500 cells per well into CellCarrier-96 spherical ULA / CS, centrifuge at 1000 rpm for 5 minutes, place in a 2.5L anaerobic culture bag, and simultaneously place a 2.5L microaerophilic gas generator bag in the bag. Incubate in a 37°C incubator with 5% CO2 for 10 days, changing the medium and replacing the gas generator bag every 2 days.

[0020] The supernatant was collected. After 10 days of cell culture, the supernatant from the 96-well plate was aspirated, and HGF, oncostatin-M, B27, and dexamethasone were added to the HCM medium. The mixture was gently pipetted to obtain the fourth induction medium. The final concentrations of HGF, oncostatin-M, and dexamethasone in the fourth induction medium were 10 ng / ml, 20 ng / ml, 1%, and 100 nM, respectively. The cells were then cultured in the fourth induction medium at 37°C with 5% CO2, and the medium was changed every 2 days, with the supernatant collected. After induction, HE cells exhibited a spherical or ellipsoidal three-dimensional structure under a light microscope. The edges were irregular, and granular cells were visible. The cytoplasm was abundant, and the cell spheroids slowly increased in size with increasing culture time (e.g., ...). Figure 13 (As shown). ELISA was used to detect ALB protein secretion in the organoid supernatant. Results (i.e., post-induction results) are shown below. Figure 14 As shown, the detection of ALB secretion in the supernatant of the surface organ and its slow increase over time indicate that organoid induction was successful.

[0021] In summary, by means of the above-mentioned technical solutions of the present invention, the human amniotic mesenchymal stem cell induction culture medium and induction method for directed differentiation into liver organoids of the present invention utilizes human amniotic mesenchymal stem cells with advantages such as strong proliferative capacity, ease of extraction and culture, no ethical restrictions, no immune rejection, no tumorigenicity, and multi-lineage differentiation potential. The induction method for directed differentiation of human amniotic mesenchymal stem cells into liver organoids of the present invention avoids the tumorigenicity problem that exists in other methods such as induced pluripotent stem cell induction, making it more suitable for industrialization and clinical application. The induction culture medium and induction method for directed differentiation of human amniotic mesenchymal stem cells into liver organoids of the present invention use simple components and have low technical requirements, thereby greatly increasing the feasibility of the experiment and facilitating industrialization.

[0022] It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solution of this invention. These modifications and improvements should also be considered within the scope of protection of this invention, and will not affect the effectiveness of the invention or the practicality of the patent. The scope of protection claimed in this application shall be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A culture medium for the directed differentiation of human amniotic mesenchymal stem cells into liver organoids, characterized in that, include: First induction medium: composed of fetal bovine serum, activin A, B27 and RPMI 1640 medium; The second induction medium was a mixture of BMP4, bFGF, B27 and RPMI 1640 medium. The third induction medium is a mixture of HGF, oncostatin-M, B27, dexamethasone, and RPMI 1640 medium. The fourth induction medium is a mixture of HGF, oncostatin-M, B27, dexamethasone, and HCM medium.

2. A method for culturing a medium obtained according to claim 1, characterized in that, Includes the following steps: S1. Take third-generation human amniotic mesenchymal stem cells and seed them into culture plates for expansion culture until the cell growth density reaches 75%–85%. S2. Discard the culture supernatant from step S1, add the first induction medium, and culture under normoxic cell culture standard conditions for 5–7 days. S3. Discard the culture supernatant from step S2, add the second induction medium, and culture under hypoxic cell culture conditions for 5–7 days. S4. Discard the culture supernatant from step S3, add trypsin to digest the cells, seed them into a culture carrier at a certain density, resuspend the cells in the third induction medium and centrifuge, and culture under hypoxic cell culture conditions for 8–12 days. S5. After the culture in step S4 is completed, discard the culture supernatant, add the fourth induction medium, and continue to culture under normoxic cell culture standard conditions to finally obtain functionally mature liver organoids.

3. The induction method according to claim 2, characterized in that, In the first induction medium, the final concentration of fetal bovine serum is 0.3%–0.7%, the final concentration of activin A is 90–110 ng / mL, and the final concentration of B27 is 1×; in the second induction medium, the final concentration of BMP4 is 15–25 ng / mL, the final concentration of bFGF is 90–110 ng / mL, and the final concentration of B27 is 1×; in the third induction medium, the final concentration of HGF is 8–12 ng / mL, the final concentration of oncostatin-M is 18–22 ng / mL, the final concentration of B27 is 1×, and the final concentration of dexamethasone is 90–110 nM; in the fourth induction medium, the final concentration of HGF is 8–12 ng / mL, the final concentration of oncostatin-M is 18–22 ng / mL, the final concentration of B27 is 1×, and the final concentration of dexamethasone is 90–110 nM.

4. The induction method according to claim 3, characterized in that, In the first induction medium, the final concentration of fetal bovine serum was 0.5%, the final concentration of activin A was 100 ng / mL, and the final concentration of B27 was 1×; in the second induction medium, the final concentration of BMP4 was 20 ng / mL, the final concentration of bFGF was 100 ng / mL, and the final concentration of B27 was 1×; in the third induction medium, the final concentration of HGF was 10 ng / mL, the final concentration of oncostatin-M was 20 ng / mL, the final concentration of B27 was 1×, and the final concentration of dexamethasone was 100 nM; in the fourth induction medium, the final concentration of HGF was 10 ng / mL, the final concentration of oncostatin-M was 20 ng / mL, the final concentration of B27 was 1×, and the final concentration of dexamethasone was 100 nM.

5. The induction method according to claim 4, characterized in that, The culture medium replacement times for each step are as follows: During the culture period of step S2, replace with fresh first induction medium once; during the culture period of step S3, replace with fresh second induction medium once; during the culture period of step S4, replace with fresh third induction medium every 2 days; during the culture period of step S5, replace with fresh fourth induction medium every 2 days.

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