Liver stem cells obtained by directional induced differentiation of mesenchymal stem cells and preparation method and application of liver stem cells
Through the method of directed induction and differentiation of mesenchymal stem cells, liver stem cells were prepared, which solved the problem of poor targeting in the prior art and achieved effective treatment of cirrhosis.
Patent Information
- Application Number
- CN202510582745.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-08-08
AI Technical Summary
Existing mesenchymal stem cells have poor targeting in the treatment of cirrhosis and have no obvious treatment effect.
Hepatic stem cells were prepared by culturing mesenchymal stem cells in induction medium for 5 to 7 days, then cultured in differentiation medium for 3 to 6 days, and then cultured in mature medium for 3 to 6 days, and filtered, centrifuged and dried.
The directional transformation of mesenchymal stem cells into hepatic stem cells is achieved, providing an effective means to treat cirrhosis without side effects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of cell culture technology, and in particular to a liver stem cell differentiated by directed induction of mesenchymal stem cells, and a preparation method and application thereof. Background Art
[0002] Cirrhosis is an irreversible fibrotic disease caused by chronic liver damage. Existing treatments include medication, lifestyle adjustments, and, in severe cases, liver transplantation. However, these treatments have limitations in improving liver function and reversing liver fibrosis.
[0003] Mesenchymal stem cells (MSCs) possess differentiation potential and immunomodulatory functions, capable of differentiating into functional hepatocytes to replace necrotic or diseased cells. However, existing MSCs have poor targeting properties and are ineffective in treating liver cirrhosis. Therefore, there is a need for drugs with better targeting and effective treatment for liver cirrhosis to address these shortcomings. Summary of the Invention
[0004] The purpose of the present invention is to provide a liver stem cell differentiated by directed induction of mesenchymal stem cells and a preparation method and application thereof.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The present invention provides a method for inducing the directed differentiation of mesenchymal stem cells into hepatic stem cells, comprising the following steps:
[0007] (1) Mesenchymal stem cells were inoculated into an induction medium and cultured for 5 to 7 days to obtain intermediate cells 1;
[0008] (2) Inoculating intermediate cell 1 into differentiation medium and culturing for 3 to 6 days to obtain intermediate cell 2;
[0009] (3) The intermediate cells 2 are inoculated into a maturation culture medium, cultured for 3 to 6 days, filtered, centrifuged, and the precipitate is collected and dried to obtain liver stem cells.
[0010] Preferably, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells.
[0011] Preferably, the induction medium is based on RPMI1640 medium and further includes the following components at the following concentrations:
[0012] Hepatocyte growth factor 50-100 μg / mL, β-nerve growth factor 10-18 ng / mL, hepatocyte nuclear factor 420-40 μg / mL, vascular endothelial growth factor 8-16 ng / mL, activin A3-12 ng / mL.
[0013] Preferably, the differentiation medium is based on DMEM / F12 medium and further includes the following components at the following concentrations:
[0014] Hepatocyte growth factor 50-100 μg / mL, hepatocyte nuclear factor 4 20-40 μg / mL, hesperidin 30-50 μg / mL, adenylate cyclase activator 2-8 ng / mL, glutamate 10-20 μg / mL, penicillin 50-100 U / mL, and streptomycin 50-100 U / mL.
[0015] Preferably, the maturation medium is based on DMEM / F12 medium and further includes the following components at the following concentrations:
[0016] Hepatocyte growth factor 30-50 μg / mL, hepatocyte nuclear factor 4 10-20 μg / mL, dexamethasone 3-8 μg / mL, penicillin 50-100 U / mL, streptomycin 50-100 U / mL, vitamin C 10-20 μg / mL.
[0017] Preferably, the culture temperature in any one of steps (1) to (3) is independently 20 to 30° C., and the oxygen concentration in the culture is independently 3 to 10%.
[0018] Preferably, the filtration is ultrafiltration, the pressure during ultrafiltration is 0.3-0.8 MPa, and the pore size of the filter membrane during ultrafiltration is 2-20 nm.
[0019] Preferably, the centrifugal speed is 20000-30000 rpm, and the centrifugal time is 8-10 min;
[0020] The drying is freeze drying, and the freeze drying method is: first freezing at a temperature of -5 to -8°C for 2 to 3 hours; then freezing at a temperature of -30 to -40°C and a vacuum degree of 10 to 20 Pa for 2 to 3 hours; and finally freezing at a temperature of -40 to -50°C and a vacuum degree of 10 to 20 Pa for 3 to 5 hours.
[0021] The present invention also provides liver stem cells prepared according to the method.
[0022] The present invention also provides the use of the liver stem cells in preparing medicine for treating liver cirrhosis.
[0023] The present invention provides a liver stem cell derived from mesenchymal stem cells through directional induction differentiation, and a preparation method and application thereof. The induction method of the present invention comprises the following steps: (1) inoculating mesenchymal stem cells in an induction medium and culturing for 5 to 7 days to obtain intermediate cells 1; (2) inoculating intermediate cells 1 in a differentiation medium and culturing for 3 to 6 days to obtain intermediate cells 2; (3) inoculating intermediate cells 2 in a maturation medium and culturing for 3 to 6 days, filtering, centrifuging, collecting precipitates, and drying to obtain liver stem cells. The method of the present invention first induces mesenchymal stem cells to transform into liver cells, then transfers them to a differentiation medium and begins to differentiate into liver stem cells. Finally, the differentiated liver stem cells are placed in a maturation medium to allow the undifferentiated cells to continue to differentiate into liver stem cells, and the differentiated liver stem cells are expanded and cultured. Ultimately, the directional transformation of mesenchymal stem cells into liver stem cells can be achieved. The liver stem cells obtained by the present invention can be used to treat liver cirrhosis with good efficacy and no side effects, providing a basis for the treatment of liver cirrhosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 The transformation of umbilical cord mesenchymal stem cells into liver stem cells;
[0025] Figure 2 The transformation of bone marrow mesenchymal stem cells into liver stem cells;
[0026] Figure 3 The transformation of adipose-derived mesenchymal stem cells into liver stem cells. DETAILED DESCRIPTION
[0027] The present invention provides a method for inducing the directed differentiation of mesenchymal stem cells into hepatic stem cells, comprising the following steps:
[0028] (1) Mesenchymal stem cells were inoculated into an induction medium and cultured for 5 to 7 days to obtain intermediate cells 1;
[0029] (2) Inoculating intermediate cell 1 into differentiation medium and culturing for 3 to 6 days to obtain intermediate cell 2;
[0030] (3) The intermediate cells 2 are inoculated into a maturation culture medium, cultured for 3 to 6 days, filtered, centrifuged, and the precipitate is collected and dried to obtain liver stem cells.
[0031] In the present invention, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells.
[0032] In the present invention, the induction medium is based on RPMI1640 medium and further includes the following components at the following concentrations:
[0033] Hepatocyte growth factor 50-100 μg / mL, preferably 60-90 μg / mL, more preferably 75 μg / mL;
[0034] β-nerve growth factor 10-18 ng / mL, preferably 12-16 ng / mL, more preferably 14 ng / mL;
[0035] Hepatocyte nuclear factor 420-40 μg / mL, preferably 25-35 μg / mL, more preferably 30 μg / mL;
[0036] Vascular endothelial growth factor 8-16 ng / mL, preferably 10-14 ng / mL, more preferably 12 ng / mL;
[0037] Activin A is 3 to 12 ng / mL, preferably 5 to 10 ng / mL, and more preferably 8 ng / mL.
[0038] In the present invention, the differentiation medium is based on DMEM / F12 medium and further includes the following components at the following concentrations:
[0039] Hepatocyte growth factor 50-100 μg / mL, preferably 60-90 μg / mL, more preferably 75 μg / mL;
[0040] Hepatocyte nuclear factor 420-40 μg / mL, preferably 25-35 μg / mL, more preferably 30 μg / mL;
[0041] Hesperidin 30-50 μg / mL, preferably 35-45 μg / mL, more preferably 40 μg / mL;
[0042] Adenylate cyclase activator 2-8 ng / mL, preferably 4-6 ng / mL, more preferably 5 ng / mL;
[0043] Glutamic acid 10-20 μg / mL, preferably 12-18 μg / mL, more preferably 15 μg / mL;
[0044] Penicillin 50-100 U / mL, preferably 60-90 U / mL, more preferably 75 U / mL;
[0045] Streptomycin is 50 to 100 U / mL, preferably 60 to 90 U / mL, and more preferably 75 U / mL.
[0046] In the present invention, the maturation medium is based on DMEM / F12 medium and further includes the following components at the following concentrations:
[0047] Hepatocyte growth factor 30-50 μg / mL, preferably 35-45 μg / mL, more preferably 40 μg / mL;
[0048] Hepatocyte nuclear factor 410-20 μg / mL, preferably 12-18 μg / mL, more preferably 15 μg / mL;
[0049] Dexamethasone 3-8 μg / mL, preferably 4-7 μg / mL, more preferably 6 μg / mL;
[0050] Penicillin 50-100 U / mL, preferably 60-90 U / mL, more preferably 75 U / mL;
[0051] Streptomycin 50-100 U / mL, preferably 60-90 U / mL, more preferably 75 U / mL;
[0052] Vitamin C 10-20 μg / mL, preferably 12-18 μg / mL, more preferably 15 μg / mL.
[0053] In the present invention, the culture temperature in any one of steps (1) to (3) is independently 20 to 30° C., preferably 25° C.; the oxygen concentration in the culture is independently 3 to 10%, preferably 6%.
[0054] In the present invention, the filtration is ultrafiltration, the pressure during ultrafiltration is 0.3-0.8 MPa, preferably 0.5 MPa, and the pore size of the filter membrane during ultrafiltration is 2-20 nm, preferably 10 nm.
[0055] In the present invention, the centrifugal speed is 20000-30000 rpm, preferably 25000 rpm, and the centrifugal time is 8-10 min, preferably 9 min;
[0056] The drying is freeze drying, and the freeze drying method is: first freezing at a temperature of -5 to -8°C for 2 to 3 hours; then freezing at a temperature of -30 to -40°C and a vacuum degree of 10 to 20 Pa for 2 to 3 hours; and finally freezing at a temperature of -40 to -50°C and a vacuum degree of 10 to 20 Pa for 3 to 5 hours.
[0057] Preferably, the mixture is first frozen at -6°C for 2.5 hours; then frozen at -35°C and a vacuum degree of 15 Pa for 2.5 hours; and finally frozen at -45°C and a vacuum degree of 15 Pa for 4 hours.
[0058] The present invention also provides liver stem cells prepared according to the method.
[0059] The present invention also provides the use of the liver stem cells in preparing medicine for treating liver cirrhosis.
[0060] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0061] Example 1
[0062] Preparation of induction medium: Based on RPMI1640 medium, it also includes the following components at the following concentrations: hepatocyte growth factor (HGF) 50 μg / mL, β-nerve growth factor (β-NGF) 15 ng / mL, hepatocyte nuclear factor 4 (HNF4) 40 μg / mL, vascular endothelial growth factor (VEGF) 12 ng / mL, and activin A 10 ng / mL.
[0063] Preparation of differentiation medium: Based on DMEM / F12 medium, the following components were also added: HGF 100 μg / mL, HNF4 20 μg / mL, hesperidin 30 μg / mL, adenylate cyclase activator (cAMP) 8 ng / mL, glutamate 15 μg / mL, penicillin 80 U / mL, and streptomycin 80 U / mL.
[0064] Preparation of maturation medium: Based on DMEM / F12 medium, the following components are also included: HGF 50 μg / mL, HNF4 15 μg / mL, dexamethasone 8 μg / mL, penicillin 50 U / mL, streptomycin 100 U / mL, and vitamin C 15 μg / mL.
[0065] Umbilical cord mesenchymal stem cells were resuspended in RPMI1640 medium and diluted to 1×10 6 Cells were inoculated into induction medium at 30°C and 5% oxygen for 5 days to obtain intermediate cells 1. Intermediate cells 1 were inoculated into differentiation medium and cultured at 30°C and 8% oxygen for 5 days to obtain intermediate cells 2. Intermediate cells 2 were inoculated into maturation medium and cultured at 30°C and 10% oxygen for 5 days. The cells were then placed in an ultrafiltration tube and ultrafiltration was performed at a pressure of 0.5 MPa and a 10 nm pore size. After ultrafiltration, the cells were centrifuged at 20,000 rpm for 10 minutes. The precipitate was collected and frozen at -6°C for 2 hours, then at -40°C under a vacuum of 10 Pa for 3 hours, and finally at -45°C under a vacuum of 10 Pa for 4 hours to obtain hepatic stem cells.
[0066] The supernatants of cells differentiated in differentiation medium for 2 days and cells cultured in maturation medium for 2 days were collected during the culture process to detect the secretion of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and urea nitrogen in the cell supernatants.
[0067] Aspartate aminotransferase (AST), alanine aminotransferase (ALT) and urea nitrogen are hepatocyte-specific biochemical indicators to evaluate the degree of transformation of umbilical cord mesenchymal stem cells into hepatic stem cells. Figure 1 shown.
[0068] Figure 1 It can be seen that after 2 days of culture in differentiation medium, the levels of AST, ALT, and urea nitrogen were detected in the cell supernatant, indicating that umbilical cord mesenchymal stem cells transformed into hepatic stem cells. After 2 days of culture in maturation medium, the levels of AST, ALT, and urea nitrogen in the cell supernatant increased compared to those in differentiation medium, indicating that more umbilical cord mesenchymal stem cells differentiated into hepatic stem cells.
[0069] Example 2
[0070] Preparation of induction medium: Based on RPMI1640 medium, the following components are also included: HGF 100 μg / mL, β-NGF 10 ng / mL, HNF4 25 μg / mL, VEGF 8 ng / mL, Activin A 12 ng / mL.
[0071] Preparation of differentiation medium: Based on DMEM / F12 medium, the following components are also included: HGF 80 μg / mL, HNF4 40 μg / mL, hesperidin 35 μg / mL, adenylate cyclase activator (cAMP) 6 ng / mL, glutamate 10 μg / mL, penicillin 50 U / mL, and streptomycin 100 U / mL.
[0072] Preparation of maturation medium: Based on DMEM / F12 medium, the following components are also added: HGF 30 μg / mL, HNF4 20 μg / mL, dexamethasone 3 μg / mL, penicillin 100 U / mL, streptomycin 50 U / mL, and vitamin C 20 μg / mL.
[0073] Bone marrow mesenchymal stem cells were resuspended in RPMI1640 medium and diluted to 1×10 7Cells were inoculated into induction medium at 20°C and 3% oxygen for 7 days to obtain intermediate cells 1. Intermediate cells 1 were inoculated into differentiation medium and cultured at 20°C and 6% oxygen for 5 days to obtain intermediate cells 2. Intermediate cells 2 were inoculated into maturation medium and cultured at 20°C and 8% oxygen for 5 days. The cells were then placed in an ultrafiltration tube and ultrafiltration was performed at a pressure of 0.5 MPa and a 10 nm pore size. After ultrafiltration, the cells were centrifuged at 20,000 rpm for 10 minutes. The precipitate was collected and frozen at -5°C for 3 hours, then at -30°C under a vacuum of 20 Pa for 3 hours, and finally at -40°C under a vacuum of 20 Pa for 5 hours to obtain hepatic stem cells.
[0074] The supernatant of cells cultured in differentiation medium for 2 days and in maturation medium for 2 days were collected during the culture process to detect the secretion of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and urea nitrogen in the cell supernatant. Figure 2 shown.
[0075] Figure 2 The results showed that after 2 days of culture in differentiation medium, the levels of AST, ALT, and urea nitrogen were detected in the cell supernatant, indicating that bone marrow mesenchymal stem cells were transforming into hepatic stem cells. After 2 days of culture in maturation medium, the levels of AST, ALT, and urea nitrogen in the cell supernatant increased compared to those in differentiation medium, indicating that more bone marrow mesenchymal stem cells were differentiating into hepatic stem cells.
[0076] Example 3
[0077] Preparation of induction medium: Based on RPMI1640 medium, the following components are also included: HGF 80 μg / mL, β-NGF 18 ng / mL, HNF4 20 μg / mL, VEGF 16 ng / mL, Activin A 3 ng / mL.
[0078] Preparation of differentiation medium: Based on DMEM / F12 medium, the following components were also added: HGF 50 μg / mL, HNF4 30 μg / mL, hesperidin 50 μg / mL, adenylate cyclase activator cAMP 2 ng / mL, glutamate 20 μg / mL, penicillin 80 U / mL, and streptomycin 100 U / mL.
[0079] Preparation of maturation medium: Based on DMEM / F12 medium, the following components are also included: HGF 50 μg / mL, HNF4 10 μg / mL, dexamethasone 5 μg / mL, penicillin 80 U / mL, streptomycin 80 U / mL, and vitamin C 10 μg / mL.
[0080] Adipose-derived mesenchymal stem cells were resuspended in RPMI1640 medium and diluted to 1×10 7 Cells were inoculated into induction medium at 25°C and 3% oxygen for 5 days to obtain intermediate cells 1. Intermediate cells 1 were inoculated into differentiation medium and cultured at 25°C and 5% oxygen for 5 days to obtain intermediate cells 2. Intermediate cells 2 were inoculated into maturation medium and cultured at 25°C and 7% oxygen for 5 days. The cells were then placed in an ultrafiltration tube and ultrafiltration was performed at a pressure of 0.3 MPa and a 10 nm pore size. After ultrafiltration, the cells were centrifuged at 20,000 rpm for 10 minutes. The precipitate was collected and frozen at -8°C for 2 hours, then at -35°C under a vacuum of 10 Pa for 2 hours, and finally at -50°C under a vacuum of 10 Pa for 4 hours to obtain hepatic stem cells.
[0081] The supernatant of cells cultured in differentiation medium for 2 days and in maturation medium for 2 days were collected during the culture process to detect the secretion of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and urea nitrogen in the cell supernatant. Figure 3 shown.
[0082] Figure 3 The results showed that after 2 days of culture in differentiation medium, the levels of AST, ALT, and urea nitrogen were detected in the cell supernatant, indicating that adipose-derived mesenchymal stem cells were transforming into hepatic stem cells. After 2 days of culture in maturation medium, the levels of AST, ALT, and urea nitrogen in the cell supernatant increased compared to those in differentiation medium, indicating that more adipose-derived mesenchymal stem cells were differentiating into hepatic stem cells.
[0083] Experimental Example 1
[0084] Fifty rats were selected, 10 of which served as blank controls, and the remaining 40 were treated with a cirrhosis model. The modeling method was as follows: In the first week, 0.35 g / L sodium phenobarbital solution was added to the drinking water for induction. From the second to third weeks, rats were subcutaneously injected with 40% and 50% CCl₄ rapeseed oil solutions (40 and 50 mL CCl₄ solutions, respectively, diluted to 100 mL with rapeseed oil) at a volume of 3 mL / kg three times a week. From the fourth to the 14th week, rats were subcutaneously injected with 50% CCl₄ rapeseed oil solution (60 mL CCl₄ solution, diluted to 100 mL with rapeseed oil) at a volume of 3 mL / kg three times a week. From the second to the 14th week, 10% edible alcohol was used as the sole drinking water. At the end of the 14th week, rats with sparse, dull hair, black stools, decreased appetite, decreased activity, and sluggish reflexes, which showed significant differences from the blank control rats, were considered successful models. 32 rats with successful modeling were selected and randomly divided into 4 groups, 8 rats in each group, namely Example 1 group, Example 2 group, Example 3 group and control group.
[0085] Rats in the Example 1 group were gavaged with Example 1 liver stem cells (20 mg / kg) once daily; rats in the Example 2 group were gavaged with Example 2 liver stem cells (20 mg / kg) once daily; and rats in the Example 3 group were gavaged with Example 3 liver stem cells (20 mg / kg) once daily. Rats in the control group were gavaged with 20 mg / kg of Sunflower Liver Protection Tablets once daily. Rats in the blank control group were gavaged with normal saline. This gavage was continued for two weeks.
[0086] After the last administration, the mice were fasted but not watered for 24 h, anesthetized with an intraperitoneal injection of 50 mg / kg sodium pentobarbital, 10 mL of blood was collected through the abdominal aorta, and the serum was separated for detection.
[0087] The levels of AST and ALT in serum were determined by the Reitman method and the lactate dehydrogenase rate method, respectively. The results are shown in Table 1.
[0088] Table 1 Effects of different drugs on AST and ALT levels in cirrhotic rats
[0089] Group ALT(U / L) AST(U / L) Normal control group 37.6 72.5 control group 50.36 121.37 Example 1 group 52.47 116.57 Example 2 group 49.85 123.47 Example 3 group 51.37 116.58
[0090] Table 1 shows that the effects of gavage on AST and ALT levels in rat serum of cirrhotic rats using the liver stem cells prepared using the methods of Examples 1, 2, and 3 were substantially consistent with the effects of Sunflower Liver Protection Tablets. This demonstrates that Examples 1 to 3 of the present invention can be used to induce liver stem cells targeted to hepatocytes.
[0091] The levels of interleukin-6 (IL-6) and tumor necrosis factor (TNF-α) in the serum of rats in each group were determined by ELISA. The test results are shown in Table 2.
[0092] Table 2 Effects of different drugs on inflammatory factors in the serum of cirrhotic rats
[0093] Group IL-6 (pg / mL) TNF-α (pg / mL) Normal control group 72.18 20.59 control group 126.34 35.69 Example 1 group 134.85 34.55 Example 2 group 124.37 32.58 Example 3 group 125.46 31.41
[0094] Table 2 shows that the effects of gavage on inflammatory factors in rat serum of cirrhotic rats using the liver stem cells prepared by the methods of Examples 1, 2, and 3 were substantially consistent with the effects of Sunflower Liver Protection Tablets, indicating that Examples 1 to 3 of the present invention can induce liver stem cells targeted to hepatocytes.
[0095] The levels of type III procollagen (PCⅢ), type IV collagen (IV-C) and hyaluronidase (HA) in serum were determined by radioimmunoassay. The results are shown in Table 3.
[0096] Table 3 Effects of different drugs on serum liver fibrosis
[0097] Group PCⅢ(ng / mL) IV-C (ng / mL) HA (ng / mL) Normal control group 1.31 2.09 5.27 control group 3.16 5.47 12.68 Example 1 group 2.98 6.04 11.69 Example 2 group 3.05 5.68 12.13 Example 3 group 2.86 5.21 11.64
[0098] Table 3 shows that the effects of liver stem cells prepared by the methods of Examples 1, 2 and 3 on PCⅢ, IV-C and HA in rat serum after oral administration to cirrhotic rats were basically consistent with the effects of sunflower liver protection tablets.
[0099] Liver tissue was collected from each group of mice and immunohistochemical staining was used to determine the expression of α-smooth muscle actin (α-SMA). The results were analyzed using integrated optical density (IOD) values. A higher IOD value indicates higher α-SMA expression. The results are shown in Table 4.
[0100] Table 4 Effects of different drugs on the expression of α-SMA in liver tissue
[0101] Group IOD value Normal control group 0.90 control group 2.89 Example 1 group 2.65 Example 2 group 3.01 Example 3 group 2.73
[0102] As shown in Table 4, the effects of liver stem cells prepared by the methods of Examples 1, 2 and 3 on the expression of α-SMA in rat liver tissue after oral administration to cirrhotic rats were basically consistent with the effect of Sunflower Liver Protection Tablets.
[0103] As can be seen from the above examples, the present invention provides a liver stem cell derived from mesenchymal stem cells through directional induction differentiation, and a preparation method and application thereof. The induction method of the present invention comprises the following steps: (1) inoculating mesenchymal stem cells in an induction medium and culturing for 5 to 7 days to obtain intermediate cells 1; (2) inoculating intermediate cells 1 in a differentiation medium and culturing for 3 to 6 days to obtain intermediate cells 2; (3) inoculating intermediate cells 2 in a maturation medium and culturing for 3 to 6 days, filtering, centrifuging, collecting the precipitate, and drying to obtain liver stem cells. The method of the present invention first induces mesenchymal stem cells to transform into liver cells, then transfers them to a differentiation medium and begins to differentiate into liver stem cells. Finally, the differentiated liver stem cells are placed in a maturation medium to allow the undifferentiated cells to continue to differentiate into liver stem cells, and the differentiated liver stem cells are expanded and cultured. Ultimately, the directional transformation of mesenchymal stem cells into liver stem cells can be achieved. The liver stem cells obtained by the present invention can be used to treat liver cirrhosis with good efficacy and no side effects, providing a basis for the treatment of liver cirrhosis.
[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A method for inducing the differentiation of mesenchymal stem cells into hepatic stem cells, characterized in that: The steps include: (1) Mesenchymal stem cells were inoculated into an induction medium and cultured for 5 to 7 days to obtain intermediate cells 1; (2) Inoculating intermediate cell 1 into differentiation medium and culturing for 3 to 6 days to obtain intermediate cell 2; (3) The intermediate cells 2 are inoculated into a maturation culture medium, cultured for 3 to 6 days, filtered, centrifuged, and the precipitate is collected and dried to obtain liver stem cells.
2. The method according to claim 1, characterized in that The mesenchymal stem cells are umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells.
3. The method according to claim 2, characterized in that The induction medium is based on RPMI1640 medium and further includes the following components at the following concentrations: Hepatocyte growth factor 50-100 μg / mL, β-nerve growth factor 10-18 ng / mL, hepatocyte nuclear factor 420-40 μg / mL, vascular endothelial growth factor 8-16 ng / mL, activin A3-12 ng / mL.
4. The method according to claim 3, characterized in that The differentiation medium is based on DMEM / F12 medium and further includes the following components at the following concentrations: Hepatocyte growth factor 50-100 μg / mL, hepatocyte nuclear factor 4 20-40 μg / mL, hesperidin 30-50 μg / mL, adenylate cyclase activator 2-8 ng / mL, glutamate 10-20 μg / mL, penicillin 50-100 U / mL, and streptomycin 50-100 U / mL.
5. The method according to claim 4, characterized in that The maturation medium is based on DMEM / F12 medium and further includes the following components at the following concentrations: Hepatocyte growth factor 30-50 μg / mL, hepatocyte nuclear factor 4 10-20 μg / mL, dexamethasone 3-8 μg / mL, penicillin 50-100 U / mL, streptomycin 50-100 U / mL, vitamin C 10-20 μg / mL.
6. The method according to any one of claims 1 to 5, characterized in that The temperature of the culture in any one of steps (1) to (3) is independently 20 to 30° C., and the oxygen concentration of the culture is independently 3 to 10%.
7. The method according to claim 6, characterized in that The filtration is ultrafiltration, the pressure during ultrafiltration is 0.3-0.8 MPa, and the pore size of the filter membrane during ultrafiltration is 2-20 nm.
8. The method according to claim 7, characterized in that The centrifugal speed is 20000-30000 rpm, and the centrifugal time is 8-10 minutes; The drying is freeze drying, and the freeze drying method is: first freezing at a temperature of -5 to -8°C for 2 to 3 hours; then freezing at a temperature of -30 to -40°C and a vacuum degree of 10 to 20 Pa for 2 to 3 hours; and finally freezing at a temperature of -40 to -50°C and a vacuum degree of 10 to 20 Pa for 3 to 5 hours.
9. Hepatic stem cells prepared according to the method according to any one of claims 1 to 8.
10. Use of the hepatic stem cells according to claim 9 in the preparation of a medicament for treating liver cirrhosis.