WO2020151418A1 - 一种扩增培养人肝祖细胞的培养基及其应用 - Google Patents

一种扩增培养人肝祖细胞的培养基及其应用 Download PDF

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WO2020151418A1
WO2020151418A1 PCT/CN2019/126578 CN2019126578W WO2020151418A1 WO 2020151418 A1 WO2020151418 A1 WO 2020151418A1 CN 2019126578 W CN2019126578 W CN 2019126578W WO 2020151418 A1 WO2020151418 A1 WO 2020151418A1
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hbs
liver
cells
medium
growth factor
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PCT/CN2019/126578
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French (fr)
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李尹雄
潘廷才
陈彦
庄苑琦
杨帆
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中国科学院广州生物医药与健康研究院
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Priority to EP19912048.6A priority Critical patent/EP3916082A4/en
Priority to US17/310,193 priority patent/US20220186181A1/en
Publication of WO2020151418A1 publication Critical patent/WO2020151418A1/zh

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Definitions

  • This application relates to the field of bioengineering technology, and in particular to a medium for expanding and cultivating human hepatic progenitor cells and its application.
  • liver disease country More than 100 million people suffer from viral hepatitis, fatty liver, and liver fibrosis. Among them, about 30 million chronic hepatitis patients are high-risk groups, and some patients develop liver fibrosis progressively. , Liver cirrhosis, liver failure and liver cancer and other severe liver diseases. Every year, more than 1 million new cases of liver cirrhosis enter the decompensated stage of liver cirrhosis, about 500,000 people die of liver failure, and several thousand people die of liver failure caused by food-borne or drug-induced liver poisoning. These various factors cause Liver disease causes a huge social and family burden.
  • liver transplantation There are more than 500,000 patients waiting for liver transplantation in my country each year, and the number of liver sources that can be used for transplantation is in the hundreds to more than 1,000 cases. The extremely limited source of liver organs is far from meeting the huge and urgent clinical needs.
  • Existing clinical treatments and preclinical studies have shown that bioartificial liver dialysis and hepatocyte transplantation can help improve the microenvironment and physiological conditions of the liver and the whole body, and help the liver repair and restore liver function. This is not only possible to save toxic acute liver failure, more importantly, for patients with a certain degree of advanced liver disease, it can delay or prevent the entry of end-stage liver failure. Both of these treatments require a large number of liver cells.
  • Human primary hepatocytes are ideal seed cells, mainly obtained from livers that are not suitable for liver transplantation standards.
  • primary hepatocytes cannot be cultured and expanded in vitro, and the source is limited by the shortage of liver source donors.
  • the cutting-edge cognition of stem cell research and the emergence of new technologies have brought new opportunities for this breakthrough.
  • Human pluripotent stem cells including human embryonic stem cells (ESC) and human induced pluripotent stem cells (iPSC) have a high degree of self-proliferation ability and multidirectional
  • the differentiation potential can theoretically induce differentiation into all types of human cells, including mature hepatocytes.
  • the directional differentiation of the hepatic lineage of hPSC is to simulate the development process of the liver in vivo, and induce them into definitive endoderm cells, hepatic progenitor cells (Hepatoblasts, HBs) and mature hepatocytes in turn.
  • Hepatoblasts hepatic progenitor cells
  • HBs hepatic progenitor cells
  • hepatocyte transplantation is used to treat liver disease. It is estimated that each transplantation treatment will require 109 grade hepatocytes, while the application of bioartificial liver and drug development requires more liver cells. Therefore, it is of great significance to quickly and efficiently obtain a large number of functional hepatocytes for the treatment of liver diseases such as cell transplantation and bioartificial liver dialysis.
  • HBs has a strong proliferation ability, and has the dual potential of rapid differentiation into hepatocytes and bile duct cells. Therefore, large-scale expansion of HBs derived from hPSC-induced differentiation, rapid and stable proliferation, and maintaining a good differentiation state is an ideal method to obtain a large number of functional hepatocytes.
  • HBs can be frozen to establish a HBs bank to quickly and efficiently obtain a large number of seed cells; on the other hand, HBs can be further induced to differentiate into mature hepatocytes or bile duct cells quickly; at the same time, this method can be used for the establishment of hepatic cell bank Provide feasible technical support.
  • HBs amplification methods use trophoblast cells in the culture conditions, or use additives with unclear components such as fetal bovine serum.
  • liver cells used for cell therapy are contaminated with animal serum, protein or other cells, they often cause acute immune rejection and potential animal virus infections in clinical applications. Therefore, the hepatocytes obtained by such culture will not be suitable for clinical application, which is also a factor that should be fully considered when expanding and culturing HBs in vitro.
  • HBs amplification culture method with clear chemical composition and serum-free formula.
  • Large-scale amplification and culture of HBs will help Obtain a large number of functional hepatocytes quickly and efficiently, and apply them to liver disease treatment such as cell transplantation and bioartificial liver dialysis. It has broad application prospects and huge market value.
  • This application provides a medium for expanding and cultivating human hepatic progenitor cells and its application.
  • the medium formula has clear chemical components, each component cooperates with each other, and is synergistic. Maintaining its dryness has broad application prospects and huge market value.
  • this application provides a medium for expanding and cultivating human hepatic progenitor cells, the medium comprising a liquid basal medium, an insulin-transferrin-sodium selenite mixture, cytokines, and glycogen synthesis kinase 3 ⁇ inhibitor, Hedgehog signaling pathway activator and transforming growth factor receptor ⁇ inhibitor.
  • the medium further includes albumin.
  • the inventors have established a method for directed differentiation induction of hepatic lineage in the course of long-term scientific research and practice, analyzed the signal pathways related to the proliferation and differentiation of hepatic progenitor cells (HBs), and clarified Wnt, TGF- ⁇ , BMP and Hedgehog
  • HBs hepatic progenitor cells
  • the regulation mechanism of key signal pathways on HBs proliferation and the maintenance of hepatic progenitor properties and further use cytokines and small molecule compounds to regulate related signal pathways, comprehensively screen active components, and develop support for stable HBs proliferation while maintaining better maintenance
  • the medium formula of its liver progenitor characteristics, each component and each condition cooperate to promote each other, synergistically increase the effect, large-scale culture and expansion of HBs, can better maintain its HBs characteristics, including direct and rapid differentiation into functional Mature hepatocytes or bile duct cells, transplanted into liver injury model mice, can home to colonize liver tissue, proliferate and further differentiate into
  • the liquid basal medium includes any one of RMPI1640 cell culture medium, DMEM/F12 cell culture medium, MEM cell culture medium, DMEM cell culture medium, IMDM cell culture medium, 199 cell culture medium or F10 cell culture medium. Or a combination of at least two.
  • the albumin includes human recombinant albumin and/or bovine serum albumin.
  • the mass concentration of the albumin is 5-500 ⁇ g/mL, for example, 5 ⁇ g/mL, 20 ⁇ g/mL, 40 ⁇ g/mL, 60 ⁇ g/mL, 80 ⁇ g/mL, 100 ⁇ g/mL, 200 ⁇ g/mL, 300 ⁇ g/mL mL, 400 ⁇ g/mL or 500 ⁇ g/mL.
  • the cytokine includes bone morphogenetic growth factor, hepatocyte growth factor and epidermal growth factor.
  • the bone morphogenetic growth factor includes BMP2 and/or BMP4.
  • the mass concentration of the bone morphogenetic growth factor is 1-50ng/mL, for example, it can be 1ng/mL, 5ng/mL, 10ng/mL, 15ng/mL, 20ng/mL, 25ng/mL, 30ng/mL , 35ng/mL, 40ng/mL, 45ng/mL or 50ng/mL.
  • the mass concentration of the hepatocyte growth factor is 1-100ng/mL, for example, it can be 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng/mL, 80ng/mL, 90ng/mL or 100ng/mL.
  • the mass concentration of the epidermal growth factor is 1-200ng/mL, for example, it can be 1ng/mL, 10ng/mL, 20ng/mL, 30ng/mL, 40ng/mL, 50ng/mL, 60ng/mL, 70ng /mL, 80ng/mL, 90ng/mL, 100ng/mL, 120ng/mL, 140ng/mL, 160ng/mL, 180ng/mL or 200ng/mL.
  • the volume percentage of the insulin-transferrin-sodium selenite mixture is 0.1-10%, for example, it can be 0.1%, 0.2%, 0.4%, 0.5%, 0.6%, 0.8%, 1%. %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%.
  • the glycogen synthesis kinase 3 ⁇ inhibitor includes CHIR99021 and/or CHIR98014.
  • the molar concentration of the glycogen synthesis kinase 3 ⁇ inhibitor is 10 nM-100 ⁇ M, for example, it can be 10 nM, 100 nM, 30 ⁇ M, 40 ⁇ M, 50 ⁇ M, 60 ⁇ M, 70 ⁇ M, 80 ⁇ M, 90 ⁇ M or 100 ⁇ M.
  • the transforming growth factor receptor ⁇ inhibitor includes any one or a combination of at least two of A8301, SB431542 or E-616452.
  • the molar concentration of the transforming growth factor receptor beta inhibitor is 50 nM-50 ⁇ M, for example, it can be 50 nM, 100 nM, 10 ⁇ M, 20 ⁇ M, 30 ⁇ M, 40 ⁇ M or 50 ⁇ M.
  • the Hedgehog signaling pathway activator includes SAG and/or Purmorphaminede.
  • the molar concentration of the Hedgehog signaling pathway activator is 10 nM-50 ⁇ M, for example, it can be 10 nM, 100 nM, 10 ⁇ M, 20 ⁇ M, 30 ⁇ M, 40 ⁇ M or 50 ⁇ M.
  • the GSK3 ⁇ inhibitor can be CHIR99021 or other small molecule or protein GSK3 ⁇ inhibitor.
  • the TGF- ⁇ receptor inhibitor can be A8301, SB431542 and E-616452, or other TGF- ⁇ receptor inhibitors.
  • the Hedgehog signal pathway activator can be other Hedgehog signal pathway activators such as SAG or Purmorphaminede.
  • the culture medium includes: liquid basal medium, 5-500 ⁇ g/mL human recombinant albumin or bovine serum albumin; 0.1%-10% (volume percentage) insulin-transferrin-sodium selenite mixture Solution (ITS); 10nM-10 ⁇ M glycogen synthesis kinase 3 ⁇ (GSK3 ⁇ ) inhibitor; 50nM-50 ⁇ M transforming growth factor ⁇ (TGF- ⁇ ) receptor inhibitor; 1-50ng/mL bone morphogenetic growth factor ( BMP2 or BMP4); 1-100ng/mL hepatocyte growth factor (HGF); 1-200ng/mL epidermal growth factor (EGF); 10nM-50 ⁇ M Hedgehog signaling pathway activator.
  • liquid basal medium 5-500 ⁇ g/mL human recombinant albumin or bovine serum albumin
  • ITS insulin-transferrin-sodium selenite mixture Solution
  • GSK3 ⁇ insulin-transferrin-sodium selenite mixture Solution
  • GSK3 ⁇ insulin-transfer
  • this application provides a method for long-term in vitro expansion and culture of human hepatic progenitor cells (HBs), using the medium described in the first aspect.
  • the method includes the following steps:
  • the source of the HBs in step (1) includes any one or a combination of at least two of human pluripotent stem cells hPSC, liver stem cells, hepatocytes or oval cells.
  • the HBs described in step 1) include HBs obtained from human pluripotent stem cells hPSC induced differentiation, and HBs derived from other sources, including liver stem cells, hepatocytes, oval cells, etc. derived from human adult liver tissue.
  • the method of obtaining in step (1) includes flow cytometry sorting.
  • a method for long-term in vitro expansion and culture of human hepatic progenitor cells HBs specifically includes the following steps:
  • the source of the HBs includes any one of human pluripotent stem cells hPSC, liver stem cells, hepatocytes or oval cells or A combination of at least two;
  • step 2 The HBs obtained in step 2) is further differentiated into functional mature hepatocytes or bile duct cells.
  • this application provides a human hepatic progenitor cell HBs, which is obtained by expansion and culture using the medium described in the first aspect or the method described in the second aspect.
  • the HBs provided in this application can be cultured in the medium for more than 50 generations, and maintain a stable phenotype and related functions of HBs, including the ability to directly and rapidly differentiate into functional mature hepatocytes or bile duct cells.
  • this application provides an application of the HBs as described in the third aspect in the preparation of transplanted cells for the treatment of liver diseases, hepatocytes used in bioartificial liver reactors, liver tissue engineering or in vitro drug screening for liver diseases.
  • This application provides a system and method for long-term expansion and cultivation of HBs in vitro and maintaining its dryness, including an expansion medium with a clear chemical composition, which is used for in vitro expansion and cultivation of human HBs.
  • This application can selectively culture and expand HBs obtained from hPSC differentiation.
  • HBs can be cultured and expanded on a large scale for more than 50 generations and maintain a stable HBs phenotype, including direct and rapid differentiation into functionally mature HBs Hepatocytes or bile duct cells, expanded HBs transplanted into liver injury model mice, can be homed and colonized in liver tissue, proliferate and further differentiate into hepatocytes expressing human ALB and bile duct cells expressing human CK19, participating in liver parenchyma The repair of tissues and the reconstruction of bile ducts can effectively restore the function of the injured liver and save the liver injury model animals.
  • This application provides a clear chemical composition and serum-free medium formulation and culture method for long-term expansion and culture of HBs induced by hPSC in vitro, which helps to quickly and efficiently obtain a large number of functional liver cells, HBs It can be cultured under this condition for more than 50 generations, and maintain stable HBs phenotype and related functions, including direct and rapid differentiation into functional mature hepatocytes or bile duct cells; HBs cultivated in this application are transplanted into liver injury model mice In vivo, it can be homed and colonized in liver tissue, participate in the repair of liver parenchymal tissue and the reconstruction of bile ducts, effectively restore the function of damaged liver, and save liver damage model animals; therefore, this invention will help to quickly and efficiently obtain a large amount of Functional liver cells are suitable for clinical liver cell transplantation applications and hepatocyte reactors in bioartificial livers.
  • Figure 1A is a flow chart of the application for inducing the directed differentiation of hPSC liver lines
  • Figure 1B shows the changes in cell morphology during the induction of HBs in this application
  • Figure 1C is an immunofluorescence identification diagram of DE cells induced to day 3 and HBs on day 10 of the application;
  • Figure 1D is a flow cytometry analysis of the expression of marker proteins at different stages in the differentiation process of HBs and the expression of the cell proliferation marker protein Ki67 in this application;
  • Figure 2A is a diagram of the application’s RT-PCR quantitative analysis of signal pathway activity related to HBs proliferation and differentiation;
  • 2B-2D are diagrams showing the influence of the signal pathways related to the regulation of small molecules on the proliferation and differentiation of HBs in this application;
  • Figure 2E is a schematic diagram of the signal pathway regulating HBs proliferation and differentiation of the application.
  • Figure 2F is a schematic diagram of screening cytokines and small molecule compounds of the application.
  • Figure 2G is a diagram showing the influence of different medium formulations of the application on the proliferation of HBs
  • Figure 2H is a cell morphology diagram showing the effects of different medium formulations of the application on the proliferation of HBs;
  • Figure 2I is a diagram showing the influence of different medium formulations of the application on the maintenance of HBs characteristics
  • Figure 2J is an analysis diagram of the influence of different medium formulations of the application on the expression of HBs-related genes
  • Figure 3A is the flow cytometric purification of HBs induced by hPSC in this application.
  • Figure 3B is a graph showing the ability of the sorted HBs of the application to form clones under expansion culture conditions
  • Fig. 3C-Fig. 3D are diagrams showing the high proliferation ability of HBs after long-term expansion in this application;
  • Figure 3E is a detection diagram of HBs marker protein expression of the application.
  • 3F-FIG. 3G are graphs showing the results of comparative analysis of HBs before and after amplification of the transcriptome of this application;
  • Figure 4A is a morphological diagram of the application of the expanded HBs differentiated into mature hepatocytes
  • Figure 4B is a diagram showing the differentiation of the amplified HBs into hepatocytes expressing marker proteins in this application;
  • 4C-FIG. 4F are the functional identification diagrams of metabolic detoxification, albumin, urea, and glycogen synthesis of hepatocytes obtained by induced differentiation in this application;
  • Figure 4G is a diagram showing the result of differentiation of HBs into bile duct epithelial cells and bile ducts of the application;
  • Figure 5A is a schematic diagram of the HBs transplantation experiment of this application.
  • Figure 5B is an analysis diagram of animal survival rate before and after HBs transplantation in this application.
  • Fig. 5C is a morphological structure diagram of liver tissue before and after cell transplantation in the pathological section analysis of the application;
  • 5D-5F are the analysis diagrams of cell homing efficiency after one week of cell transplantation in the liver tissue section analysis of this application;
  • Figure 5G is a diagram showing the differentiation of the transplanted HBs into bile duct epithelial cells and regeneration of bile ducts;
  • Figure 5H is a secretion diagram of human albumin in the serum of mice transplanted with HBs according to the application;
  • Fig. 5I is a graph of human hepatocytes 4 weeks after cell transplantation in liver tissue section analysis of the application.
  • the basic medium namely RPMI1640 medium and supplemented with 50 ⁇ g/mL human recombinant albumin, 1% (volume percentage) insulin-transferrin-sodium selenite mixture (ITS).
  • HBs expansion medium including the above-mentioned basic medium and each expansion plan combination: B 20 CEH, AB 20 CEH, AB 10 CEHS, ACEHS, ACeh; the letters in the amplification plan combination represent: A is a 50nM-50 ⁇ M transformation Growth factor ⁇ (TGF- ⁇ ) receptor inhibitor A8301, B 10 or B 20 are bone morphogenetic growth factors (BMP2 or BMP4) at 10 ng/mL or 10 ng/mL, respectively, and C is glycogen synthesis kinase at 10 nM-10 ⁇ M 3 ⁇ (GSK3 ⁇ ) inhibitor, E is 1-200ng/mL epidermal growth factor (EGF), H is 1-100ng/mL hepatocyte growth factor (HGF), S is 10nM-50 ⁇ M Hedgehog signaling pathway activator.
  • TGF- ⁇ transformation Growth factor ⁇
  • B 10 or B 20 are bone morphogenetic growth factors (BMP2 or BMP4) at 10 ng/mL or 10 ng/mL,
  • the optimized HBs amplification medium which is the above-mentioned basic medium and includes the amplification program AB 10 CEHS combination, which includes 5 ⁇ M A8301, 20ng/mL BMP4, 3 ⁇ M CHIR99021, 20ng/mL EGF, 20ng/mL HGF, 0.5 ⁇ M SAG;
  • the cells grew to 80% confluence, they were digested with Accutase enzyme and subcultured.
  • hPSC H1 or iPSC, from the Key Stem Cell Laboratory
  • hPSC was cultured on a 12-well culture plate coated with 1% Matrigel (growth factor reduced, BD Bioscience), and the medium was mTeSR1 (Stem Cell Technologies).
  • mTeSR1 Stem Cell Technologies
  • RPMI1640 contains 2% B27 (minus insulin, Invitrogen), 3 ⁇ M CHIR99021 (CHIR) and 100ng/mL Activin A for one day, then CHIR is removed and the induction is continued for two days , The cells differentiate into DE cells; then, change the medium to the hepatic progenitor-oriented differentiation medium, RPMI1640 contains 2% B27 (Invitrogen), 20ng/mL BMP2, 20ng/mL BMP4 and 30ng/mL FGF4, and induce culture for 4 days; Then change the medium, RPMI1640 contains 2% B27, 20ng/mL BMP4 and 20ng/mL HGF4, continue to induce culture for 3 days, DE cells can further differentiate into HBs.
  • FIG. 1A-1D The HBs induction process and the identification of cells at various stages are shown in Figure 1A-1D, in which the flow chart of inducing hPSC hepatic lineage differentiation (Figure 1A); cell morphology changes during HBs induction ( Figure 1B); Immunofluorescence identification of DE cells induced to day 3 and HBs on day 10 ( Figure 1C); flow cytometry analysis of the expression of marker proteins at different stages during HBs differentiation and the expression of cell proliferation marker protein Ki67 ( Figure 1D) .
  • hPSC can be induced to differentiate into HBs, and HBs has strong proliferation ability, can be expanded in a large amount under suitable culture conditions, and can be further rapidly differentiated into functionally mature hepatocytes and bile duct cells; therefore, HBs can be obtained from hPSC induction, And large-scale expansion provides a source for quickly and efficiently obtaining a large number of functional liver cells, which is an effective method to meet the large number of liver cells required for clinical liver cell transplantation treatment and liver cell reactors in bioartificial livers.
  • hepatic differentiation induction methods analyze the signal pathways related to HBs proliferation and differentiation, and clarify the regulatory mechanisms of key signal pathways such as Wnt, TGF- ⁇ , BMP and Hedgehog on the proliferation of HBs and the maintenance of hepatic progenitor properties; and Furthermore, cytokines and small molecule compounds are used to regulate related signal pathways, and a medium formula that supports the stable proliferation of HBs while maintaining its liver progenitor properties is developed.
  • RT-PCR was used to quantitatively analyze the activity of signal pathways related to HBs proliferation and differentiation; the results showed that the activities of related signal pathways such as Wnt and Hedgehog that promote HBs proliferation were down-regulated during the differentiation process ( Figure 2A) ).
  • A is 50nM-50 ⁇ M transforming growth factor ⁇ (TGF- ⁇ ) receptor inhibitor A8301
  • B 10 or B 20 is 10ng/mL or 10ng/mL, respectively Bone morphogenetic growth factor (BMP2 or BMP4)
  • C is a 10nM-10 ⁇ M glycogen synthesis kinase 3 ⁇ (GSK3 ⁇ ) inhibitor
  • E is 1-200ng/mL epidermal growth factor (EGF)
  • H 1-100ng/mL Hepatocyte growth factor (HGF)
  • S is an activator of Hedgehog signaling pathway of 10nM-50 ⁇ M;
  • the HBs obtained from hPSC differentiation were digested into single cells by Accutase (life) and resuspended in PBS containing 3% BSA, and then incubated with Ep-CAM (Milteny) and C-Kit (BD Biosciences) antibodies, and then used flow cytometry Sort Ep-CAM + /C-Kit - HBs; the HBs obtained by sorting are expanded and cultured in a culture plate coated with 1% Matrigel.
  • the medium is the optimized HBs expansion medium AB 10 CEHS, which is the basic
  • the medium RPMI1640 contains 2% B27 and 1% ITS (life), contains cytokines 20ng/mL BMP4, 20ng/mL EGF, and small molecule compounds 20ng/mL HGF, 3 ⁇ M CHIR, 5 ⁇ M A8301, 0.5 ⁇ M SAG; cells grow to When 80% is fused, it is digested with Accutase enzyme and subcultured.
  • Figures 3A-3G show the long-term expansion and identification of HBs.
  • the HBs derived from hPSC induction is purified by flow sorting (Figure 3A); the HBs obtained by the sorting have the ability to form clones under the conditions of amplification and culture ( Figure 3A).
  • Figure 3B HBs after long-term amplification still maintains a high proliferation ability ( Figure 3C- Figure 3D; detection of HBs marker protein expression (Figure 3E); transcriptome comparative analysis of HBs before and after amplification ( Figure 3F- Figure 3G) ).
  • the amplified HBs express a series of HBs marker proteins, such as AFP, HNF4, EpCAM, E-CAD, SOX9, etc.; at the same time, the results of flow cytometry analysis showed that Ki-67 and EpCAM double positive The sex rate of cells is nearly 60%, indicating that HBs has a strong proliferation ability.
  • HBs can be continuously expanded and cultured for more than 50 generations; the optimized liver expansion medium supports long-term expansion of HBs and can well maintain its hepatic progenitor characteristics; HBs after long-term expansion maintains strong proliferation ability.
  • HBs amplification culture system and method established in this application can selectively amplify the HBs induced by hPSC for a long time, and maintain its stable HBs phenotype and related functions.
  • Example 3 Induce HBs to differentiate into functionally mature hepatocytes and bile duct cells
  • HepatoZYME-SFM Gibco
  • OSM OncostatinM
  • DEX dexamethasone
  • NH4Cl 0.5mM NH4Cl
  • Figures 4A-4G show that the expanded HBs maintain the dual potential to differentiate into hepatocytes and bile duct cells, and the expanded HBs can efficiently induce differentiation into hepatocytes and express the corresponding marker proteins ALB, CK18, HNF6, E-CAD and CYP3A4 (Figure 4A- Figure 4B); hepatocytes obtained by induction of differentiation have strong metabolic detoxification, urea synthesis and other mature liver cell functions ( Figure 4C- Figure 4F); HBs differentiate into bile duct epithelial cells ( Figure 4G) ), the amplified HBs can efficiently differentiate into hepatocyte-like cells and bile duct cells, indicating that long-term expansion does not affect their differentiation ability; the hepatocytes obtained by induced differentiation have strong functions of mature liver cells such as metabolism, detoxification and urea synthesis;
  • both the expanded and unexpanded HBs can differentiate into bile duct structures in the bile duct differentiation medium and express bile duct cell marker proteins such as CK7 and CK19.
  • cytokines used for the above cell culture and induction were purchased from PeproTech, and the small molecule compounds were purchased from Selleck.
  • DMN was used to treat immunodeficient NSI mice to establish an acute liver injury model. Then, the pre-amplified and post-amplified HBs were transplanted into liver injury model mice to verify the proliferation and differentiation of the two cells in vivo and the function of repairing the damaged liver.
  • Figure 5A- Figure 5I shows the map of the transplanted HBs repair liver injury model mice, in which, the schematic diagram of the HBs transplantation experiment ( Figure 5A); the animal survival rate analysis before and after HBs transplantation (Figure 5B); the pathological section analysis of the liver tissue morphology before and after cell transplantation Structure ( Figure 5C); liver tissue section analysis of cell homing efficiency one week after cell transplantation ( Figure 5D- Figure 5F); transplanted HBs can differentiate into bile duct epithelial cells and regenerate bile ducts ( Figure 5G); detection of mice transplanted with HBs Secretion of human albumin in serum (Figure 5H); liver tissue section analysis of human hepatocytes 4 weeks after cell transplantation ( Figure 5I).
  • the two HBs before and after expansion can be homed and colonized in the recipient liver after spleen transplantation; the transplanted HBs can differentiate into hepatocytes expressing human ALB and expressing human CK19
  • the bile duct epithelial cells and participate in the regeneration and repair of liver injury parenchymal tissue and the regeneration and reconstruction of bile duct, restore liver function and improve the survival rate of liver injury model mice.
  • a medium for expanding and cultivating human hepatic progenitor cells and its application are provided.
  • the medium has a simple and reasonable formula, clear chemical composition, no serum, and each component cooperates with each other to achieve synergistic effect.
  • Long-term expansion and cultivation of HBs in vitro and maintaining its dryness will help to quickly and efficiently obtain a large number of functional hepatocytes. It is suitable for clinical hepatocyte transplantation applications and the use of hepatocyte reactors in bioartificial livers.
  • the increased life of millions of compensated/decompensated cirrhosis patients and their patients with acute liver poisoning brings hope, and has broad application prospects and huge market value.

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Abstract

提供了一种扩增培养人肝祖细胞的培养基及其应用，所述培养基配方化学成分明确，无血清，各组分相互配合，协同增效，用于体外长期扩增培养肝祖细胞并维持其干性，有助于快速高效的获取大量的具有功能的肝细胞，适宜于临床肝细胞移植应用以及生物人工肝中的肝细胞反应器使用。

Description

一种扩增培养人肝祖细胞的培养基及其应用 技术领域
本申请涉及生物工程技术领域，尤其涉及一种扩增培养人肝祖细胞的培养基及其应用。
背景技术
我国是肝病大国，有超过1亿人群患有病毒性肝炎、脂肪肝和肝纤维化等各类肝病，其中有约3000万的慢性肝炎患者是高风险人群，部分患者进行性发展为肝纤维化、肝硬化、肝衰竭和肝癌等重症肝病。每年有超过100万新发病例进入肝硬化失代偿期，50万左右死于肝衰竭，另有几千人死于食源或药源性肝中毒导致的肝衰竭，这些各类因素导致的肝脏疾病造成巨大的社会和家庭负担。
目前治疗肝衰竭的唯一有效方法就是肝脏移植。我国每年等待肝移植的病人大于50万，而能用于移植的肝脏来源数目在数百至一千余例，极其有限的肝脏器官来源远远不能满足巨大而急迫的临床需求。已有的临床治疗和临床前研究表明，生物人工肝透析和肝细胞移植，都有助于改善肝脏及全身微环境和生理状况，有利于肝脏修复并恢复肝脏功能。这不仅有可能挽救中毒性急性肝衰竭，更重要的是对于一定程度的晚期肝病患者，能延缓或阻止进入终末期肝衰竭。这两种治疗方法都需要用到大量的肝细胞。人原代肝细胞是理想的种子细胞，主要从不适合肝移植标准的肝脏中获得。但是，原代肝细胞在体外不能培养扩增，而且来源也受限于肝源供体短缺。近年来干细胞研究的前沿认知及新技术的涌现，为这个突破带来了新的契机。
人多能干细胞(Human pluripotent stem cells，hPSC)包括人胚胎干细胞(Human embryonic stem cells，ESC)和人诱导性多能干细胞(Human induced pluripotent stem cells，iPSC)，拥有的高度自我增殖能力和多向分化潜能，理论上可以诱导分化为人体所有类型的细胞，包括成熟肝细胞。hPSC的肝系定向分化就是模拟体内肝脏的发育过程，依次将其诱导为定型内胚层细胞，肝祖细胞(Hepatoblasts，HBs)和成熟肝细胞。由hPSC大量制备临床应用的肝系供体细胞，还存在一定的困难，其主要障碍在于诱导hPSC肝系定向分化的效率较低，以及难以维持所获的肝细胞在体外大量培养扩增。尤其是后者，难于获取所需要的大量肝细胞。临床上肝细胞移植治疗肝病，预计每次移植治疗需要10 9级肝细胞，而应用于生物人工肝和药物研发则需要更多的肝细胞。因此，快速高效的获取大量的具有功能的肝细胞，应用于细胞移植和生物人工肝透析等肝病治疗具有重要意义。
在体外培养条件下，分化成熟的肝细胞难以长期培养扩增，并维持其固有的细胞特性。与之相比，HBs具有较强的增殖能力，同时具有快速向肝细胞和胆管细胞分化的双潜能。因此，规模化扩增hPSC诱导分化来源的HBs，使其快速稳定增殖，并保持良好的分化状态，是获取大量具有功能的肝细胞的理想方法。一方面可以冻存HBs，建立HBs库，以便快速高效的获取大量种子细胞；另一方面可以进一步将HBs快速诱导分化为成熟的肝细胞或者胆管细胞；同时该方法可以为肝系细胞库的建立提供可行的技术支持。
近几年，有多个实验室相继报道了扩增HBs的相关研究。但是，这些研究报道大多以少数几种HBs的标志蛋白来评估扩增后的HBs的双潜能特性，而且缺乏很多相关的细胞功能评估。另外，部分HBs的扩增方法的培养条件中采用了滋养层细胞，或者使用了胎牛血清等成分不明确的添加物。然而，将用于细胞治疗的肝细胞污染有动物血清、蛋白或者其他细胞时，常在临床应用中引起急性免疫排斥反应及潜在的动物病毒感染。因此，这样培养获取的肝细胞将不适合临床应用，这也是体外扩增培养HBs时应充分考虑的因素。
目前业内尚未建立在体外长期培养扩增HBs的有效方法，其难点在于hPSC向肝系细胞分化过程和调控机制还缺乏深入的了解，以及HBs增殖和干性维持的调控机理尚未清楚。
因此，阐明hPSC肝系定向分化的调控机理，厘清HBs增殖和干性维持的调控机理，并 建立化学成分明确和无血清配方的HBs扩增培养方法，规模化扩增培养HBs，将有助于快速高效的获取大量的具有功能的肝细胞，应用于细胞移植和生物人工肝透析等肝病治疗，具有广阔的应用前景和巨大的市场价值。
发明内容
以下是对本文详细描述的主题的概述。本概述并非是为了限制权利要求的保护范围。
本申请提供一种扩增培养人肝祖细胞的培养基及其应用，所述培养基配方化学成分明确，各组分相互配合，协同增效，用于人肝祖细胞的体外扩增培养并维持其干性，具有广阔的应用前景和巨大的市场价值。
为达此目的，本申请采用以下技术方案：
第一方面，本申请提供一种扩增培养人肝祖细胞的培养基，所述培养基包括液体基础培养基、胰岛素-转铁蛋白-亚硒酸钠混合液、细胞因子、糖原合成激酶3β抑制剂、Hedgehog信号通路激活剂和转化生长因子受体β抑制剂。
优选地，所述培养基还包括白蛋白。
本申请中，发明人在长期科研实践过程中，已经建立肝系定向分化诱导方法的基础上，分析肝祖细胞(HBs)增殖和分化相关的信号通路，厘清Wnt、TGF-β、BMP和Hedgehog等关键信号通路对HBs增殖和肝祖特性维持的调控机制；并进一步用细胞因子以及小分子化合物去调控相关的信号通路，全面筛选活性组分，以开发出支持HBs稳定增殖同时较好的维持其肝祖特性的培养基配方，各组分各条件相互配合促进，协同增效，规模化培养扩增获取的HBs，能较好的维持其HBs的特性，包括可以直接快速分化为具有功能的成熟肝细胞或者胆管细胞，移植到肝损伤模型小鼠体内，可以归巢定植到肝脏组织中，增殖并进一步分化为表达人ALB的肝细胞和表达人CK19的胆管细胞，参与肝实质组织的修复以及胆管的重建，有效恢复损伤肝脏的功能，并拯救肝损伤模型动物。
优选地，所述液体基础培养基包括RMPI1640细胞培养基、DMEM/F12细胞培养基、MEM细胞培养基、DMEM细胞培养基、IMDM细胞培养基、199细胞培养基或F10细胞培养基中的任意一种或至少两种的组合。
优选地，所述白蛋白包括人重组白蛋白和/或牛血清白蛋白。
优选地，所述白蛋白的质量浓度为5-500μg/mL，例如可以是5μg/mL、20μg/mL、40μg/mL、60μg/mL、80μg/mL、100μg/mL、200μg/mL、300μg/mL、400μg/mL或500μg/mL。
优选地，所述细胞因子包括骨形态发生生长因子、肝细胞生长因子和表皮生长因子。
优选地，所述骨形态发生生长因子包括BMP2和/或BMP4。
优选地，所述骨形态发生生长因子的质量浓度为1-50ng/mL，例如可以是1ng/mL、5ng/mL、10ng/mL、15ng/mL、20ng/mL、25ng/mL、30ng/mL、35ng/mL、40ng/mL、45ng/mL或50ng/mL。
优选地，所述肝细胞生长因子的质量浓度为1-100ng/mL，例如可以是1ng/mL、10ng/mL、20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL或100ng/mL。
优选地，所述表皮生长因子的质量浓度为1-200ng/mL，例如可以是1ng/mL、10ng/mL、20ng/mL、30ng/mL、40ng/mL、50ng/mL、60ng/mL、70ng/mL、80ng/mL、90ng/mL、100ng/mL、120ng/mL、140ng/mL、160ng/mL、180ng/mL或200ng/mL。
优选地，所述胰岛素-转铁蛋白-亚硒酸钠混合液的体积百分含量为0.1-10％，例如可以是0.1％、0.2％、0.4％、0.5％、0.6％、0.8％、1％、2％、3％、4％、5％、6％、7％、8％、9％或10％。
优选地，所述糖原合成激酶3β抑制剂包括CHIR99021和/或CHIR98014。
优选地，所述糖原合成激酶3β抑制剂的摩尔浓度为10nM-100μM，例如可以是10nM、100nM、30μM、40μM、50μM、60μM、70μM、80μM、90μM或100μM。
优选地，所述转化生长因子受体β抑制剂包括A8301、SB431542或E-616452中的任意一种或至少两种的组合。
优选地，所述转化生长因子受体β抑制剂的摩尔浓度为50nM-50μM，例如可以是50nM、100nM、10μM、20μM、30μM、40μM或50μM。
优选地，所述Hedgehog信号通路激活剂包括SAG和/或Purmorphaminede。
优选地，所述Hedgehog信号通路激活剂的摩尔浓度为10nM-50μM，例如可以是10nM、100nM、10μM、20μM、30μM、40μM或50μM。
所述的GSK3β抑制剂可以是CHIR99021或者其他小分子或者蛋白的GSK3β抑制剂。
所述的TGF-β受体抑制剂可以是A8301、SB431542和E-616452，或者其他TGF-β受体抑制剂。
所述的Hedgehog信号通路激活剂可以是SAG或者Purmorphaminede等其他Hedgehog信号通路激活剂。
具体地，培养基包括：液体基础培养基，5-500μg/mL的人重组白蛋白或者牛血清白蛋白；0.1％-10％(体积百分含量)胰岛素-转铁蛋白-亚硒酸钠混合液(ITS)；10nM-10μM的糖原合成激酶3β(GSK3β)抑制剂；50nM-50μM的转化生长因子β(TGF-β)受体抑制剂；1-50ng/mL的骨形态发生生长因子(BMP2或者BMP4)；1-100ng/mL的肝细胞生长因子(HGF)；1-200ng/mL的表皮生长因子(EGF)；10nM-50μM的Hedgehog信号通路激活剂。
第二方面，本申请提供一种长期体外扩增培养人肝祖细胞(HBs)的方法，采用第一方面所述的培养基。
优选地，所述方法包括如下步骤：
(1)获取Ep-CAM +/C-kit -的HBs；
(2)采用第一方面所述培养基对步骤(1)获取的细胞进行体外培养传代；
优选地，步骤(1)所述HBs的来源包括人多能干细胞hPSC、肝脏干细胞、肝母细胞或卵圆细胞中的任意一种或至少两种的组合。
步骤1)中所述的HBs包括从人多能干细胞hPSC诱导分化获得的HBs，以及其他途径来源的HBs，包括人成体肝脏组织来源的肝脏干细胞、肝母细胞、卵圆细胞等。
优选地，步骤(1)所述获取的方法包括流式细胞术分选。
作为优选技术方案，一种长期体外扩增培养人肝祖细胞HBs的方法，具体包括如下步骤：
(1)流式细胞术分选获取Ep-CAM +/C-kit -的HBs，所述HBs的来源包括人多能干细胞hPSC、肝脏干细胞、肝母细胞或卵圆细胞中的任意一种或至少两种的组合；
(2)采用第一方面所述培养基对步骤(1)获取的细胞进行体外培养传代，获取足量的HBs；
(3)将步骤2)获得的HBs进一步分化为功能成熟的肝细胞或者胆管细胞。
第三方面，本申请提供一种人肝祖细胞HBs，采用第一方面所述培养基或第二方面所述方法扩增培养得到。
本申请提供的HBs可在所述培养基下培养超过50代，并维持稳定的HBs表型和相关功能，包括可以直接快速分化为具有功能的成熟肝细胞或者胆管细胞。
第四方面，本申请提供一种如第三方面所述的HBs在制备治疗肝脏疾病的移植细胞、生物人工肝反应器所用的肝细胞、肝脏组织工程或肝脏疾病体外药物筛选中的应用。
本申请提供了一种体外长期扩增培养HBs并维持其干性的体系和方法，包括一种化学成份明确的扩增培养基，用于人HBs的体外扩增培养。本申请可以选择性的培养扩增从hPSC分化获得的HBs，在此培养条件下，HBs可以规模化培养扩增超过50代，并维持稳定的HBs表型，包括可以直接快速分化为功能成熟的肝细胞或者胆管细胞，扩增的HBs移植到肝损伤模型小鼠体内，可以归巢定植到肝脏组织中，增殖并进一步分化为表达人ALB的肝细胞和表达人CK19的胆管细胞，参与肝实质组织的修复以及胆管的重建，有效恢复损伤肝脏的功能， 并拯救肝损伤模型动物。
与相关技术相比，本申请具有如下有益效果：
本申请提供了一种化学成分明确和无血清的培养基配方和培养方法，用于体外长期扩增培养由hPSC诱导来源的HBs，有助于快速高效的获取大量的具有功能的肝细胞，HBs可在此条件下培养超过50代，并维持稳定的HBs表型和相关功能，包括可以直接快速分化为具有功能的成熟肝细胞或者胆管细胞；本申请所培养的HBs移植到肝损伤模型小鼠体内，可以归巢定植到肝脏组织中，参与肝实质组织的修复以及胆管的重建，有效恢复损伤肝脏的功能，并拯救肝损伤模型动物；因此，该发明将有助于快速高效的获取大量的具有功能的肝细胞，适宜于临床肝细胞移植应用以及生物人工肝中的肝细胞反应器等使用。
在阅读并理解了附图和详细描述后，可以明白其他方面。
附图说明
图1A为本申请的诱导hPSC肝系定向分化的流程图；
图1B为本申请的HBs诱导过程中的细胞形态学变化；
图1C为本申请的诱导至第3天的DE细胞和第10天的HBs的免疫荧光鉴定图；
图1D为本申请的流式细胞术分析HBs分化过程中不同阶段的标志蛋白的表达以及细胞增殖标志蛋白Ki67的表达图；
图2A为本申请的RT-PCR定量分析HBs增殖与分化相关的信号通路活性图；
图2B-图2D为本申请的小分子调控相关信号通路对HBs增殖和分化的影响图；
图2E为本申请的信号通路调控HBs增殖和分化的模式图；
图2F为本申请的筛选细胞因子和小分子化合物模式图；
图2G为本申请的不同培养基配方对HBs增殖的影响图；
图2H为本申请的不同培养基配方对HBs增殖影响的细胞形态图；
图2I为本申请的不同培养基配方对HBs特性维持的影响图；
图2J为本申请的不同培养基配方对HBs相关基因表达影响的分析图；
图3A为本申请的流式分选纯化hPSC诱导来源的HBs；
图3B为本申请的分选HBs在扩增培养条件下形成克隆的能力图；
图3C-图3D为本申请的长期扩增后的HBs依然维持较高的增殖能力图；
图3E为本申请的HBs标志性蛋白表达的检测图；
图3F-图3G为本申请的转录组对比分析扩增前后的HBs结果图；
图4A为本申请的扩增后的HBs分化为成熟肝细胞的形态图；
图4B为本申请的扩增后的HBs分化为肝细胞表达标志性蛋白图；
图4C-图4F为本申请的诱导分化获取的肝细胞的代谢解毒、合成白蛋白、尿素、糖原的功能鉴定图；
图4G为本申请的HBs分化为胆管上皮细胞以及胆管结果图；
图5A为本申请的HBs移植实验示意图；
图5B为本申请的HBs移植前后的动物存活率分析图；
图5C为本申请的病理切片分析细胞移植前后肝组织的形态结构图；
图5D-图5F为本申请的肝组织切片分析细胞移植一周后的细胞归巢效率分析图；
图5G为本申请的移植的HBs分化为胆管上皮细胞，再生胆管图；
图5H为本申请的移植HBs的小鼠血清中人源白蛋白的分泌图；
图5I为本申请的肝组织切片分析细胞移植4周后的人源肝细胞图。
具体实施方式
为更进一步阐述本申请所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本申请的技术方案，但本申请并非局限在实施例范围内。
实施例1制备培养基
基础培养基，即RPMI1640培养基并补充含有50μg/mL的人重组白蛋白，1％(体积百分含量)胰岛素-转铁蛋白-亚硒酸钠混合液(ITS)。
HBs扩增培养基，包括上述基础培养基和各个扩增方案组合：B 20CEH，AB 20CEH，AB 10CEHS，ACEHS，ACEH；扩增方案组合中的字母代表：A是50nM-50μM的转化生长因子β(TGF-β)受体抑制剂A8301，B 10或者B 20分别是10ng/mL或者10ng/mL的骨形态发生生长因子(BMP2或者BMP4)，C是10nM-10μM的糖原合成激酶3β(GSK3β)抑制剂，E是1-200ng/mL的表皮生长因子(EGF)，H是1-100ng/mL的肝细胞生长因子(HGF)，S是10nM-50μM的Hedgehog信号通路激活剂。
优化的HBs扩增培养基，即上述基础培养基并包含扩增方案AB 10CEHS组合，即包括5μM A8301，20ng/mL BMP4，3μM CHIR99021，20ng/mL EGF，20ng/mL HGF，0.5μM SAG；细胞生长至80％融合时用Accutase酶进行消化并传代培养。
实施例2扩增hPSC分化获得的HBs
hPSC(H1或者iPSC，来源于干细胞重点实验室)培养于1％Matrigel(growth factor reduced，BD Bioscience)包被的12孔培养板，培养基为mTeSR1(Stem Cell Technologies)。hPSC生长至60-70％融合时更换为DE定向诱导培养基，RPMI1640包含2％B27(minus insulin，Invitrogen)，3μM CHIR99021(CHIR)和100ng/mL Activin A诱导一天，接着撤去CHIR继续诱导两天，细胞定向分化为DE细胞；然后，更换培养基为肝祖定向分化培养基，RPMI1640包含2％B27(Invitrogen)，20ng/mL BMP2，20ng/mL BMP4和30ng/mL FGF4，诱导培养4天；然后更换培养基，RPMI1640包含2％B27，20ng/mL BMP4和20ng/mL HGF4，继续诱导培养3天，DE细胞可进一步分化为HBs。
HBs的诱导过程以及相关各阶段细胞的鉴定如图1A-图1D所示，其中，诱导hPSC肝系定向分化的流程图(图1A)；HBs诱导过程中的细胞形态学变化(图1B)；免疫荧光鉴定诱导至第3天的DE细胞和第10天的HBs(图1C)；流式细胞术分析HBs分化过程中不同阶段的标志蛋白的表达以及细胞增殖标志蛋白Ki67的表达(图1D)。
hPSC可以诱导分化为HBs，而HBs具有较强的增殖能力，在合适的培养条件下可以大量扩增，且可进一步快速分化为功能成熟的肝细胞和胆管细胞；因此，从hPSC诱导获取HBs，并进行规模化扩增，为快速高效的获取大量的具有功能的肝细胞提供来源，是满足临床肝细胞移植治疗以及生物人工肝中的肝细胞反应器所需大量肝细胞的一个有效方法。
目前业内尚未建立在体外长期培养扩增HBs的有效方法，其难点在于hPSC向肝系细胞分化过程和调控机制还缺乏深入的了解，以及HBs增殖和干性维持的调控机理尚未清楚。因此，阐明hPSC肝系定向分化的调控机理，厘清HBs增殖和干性维持的调控机理，并建立化学成分明确和无血清配方的HBs扩增培养方法，规模化扩增培养HBs，将有助于快速高效的获取大量的具有功能的肝细胞，应用于细胞移植和生物人工肝透析等肝病治疗。
在已经建立肝系定向分化诱导方法的基础上，分析HBs增殖和分化相关的信号通路，厘清Wnt、TGF-β、BMP和Hedgehog等关键信号通路对HBs增殖和肝祖特性维持的调控机制；并进一步用细胞因子以及小分子化合物去调控相关的信号通路，开发出支持HBs稳定增殖同时较好的维持其肝祖特性的培养基配方。
首先，在hPSC定向分化为HBs过程中，采用RT-PCR定量分析HBs增殖与分化相关的信号通路活性；结果显示促进HBs增殖相关的Wnt、Hedgehog等相关信号通路活性在分化过程中下调(图2A)。
进一步采用小分子调控相关信号通路，分析各个信号通路对HBs增殖和分化的影响；结果显示CHIR、A8301和SAG等均能促进HBs增殖(图2B-图2C)。
进一步分析相关信号通路对HBs增殖的同时对HBs特性的影响；结果显示，CHIR促进HBs增殖的同时会抑制AFP的表达，即抑制HBs特性的维持；而A8301和SAG则能促进 AFP等肝系细胞相关基因的表达，即促进HBs特性的(图2D)。
以上结果揭示了Wnt、TGF-β、BMP和Hedgehog等相关信号通路调控HBs增殖和分化的平衡关系(图2E)。
基于此，进一步筛选小分子化合物，优化HBs扩增培养配方(图2F)；扩增培养发现，多个培养基配方都可以促进HBs增殖(图2G-图2H)；但是不同培养基配方对HBs特性维持的效果不一样，其中优化的培养基配方AB 10CEHS可以促进HBs增殖的同时能较好的维持肝祖的特性(图2I-图2J)，不同培养基配方对HBs特性维持影响的统计表见表1，扩增方案组合中的字母代表：A是50nM-50μM的转化生长因子β(TGF-β)受体抑制剂A8301，B 10或者B 20分别是10ng/mL或者10ng/mL的骨形态发生生长因子(BMP2或者BMP4)，C是10nM-10μM的糖原合成激酶3β(GSK3β)抑制剂，E是1-200ng/mL的表皮生长因子(EGF)，H是1-100ng/mL的肝细胞生长因子(HGF)，S是10nM-50μM的Hedgehog信号通路激活剂；
表1
处理 AFP + HNF4α +
B 20CEH 81.8％+/-4.2 83.4％+/-4.8
AB 20CEH 94.5％+/-3.3 96.2％+/-2.3
AB 10CEHS 96.7％+/-5.4 98.8％+/-6.0
ACEHS 77.2％+/-5.0 86.2％+/-4.5
ACEH 72.7％+/-5.2 81.5％+/-6.5
hPSC分化获得的HBs，经Accutase(life)消化成单细胞并重悬在含有3％BSA的PBS中，进一步孵育Ep-CAM(Milteny)和C-Kit(BD Biosciences)抗体，然后采用流式细胞术分选Ep-CAM +/C-Kit -的HBs；分选获取的HBs于1％Matrigel包被的培养板中进行扩增培养，培养基为优化的HBs扩增培养基AB 10CEHS，即基础培养基RPMI1640包含2％B27和1％ITS(life)，包含细胞因子20ng/mL BMP4，20ng/mL EGF，以及小分子化合物20ng/mL HGF，3μM CHIR，5μM A8301，0.5μM SAG；细胞生长至80％融合时用Accutase酶进行消化并传代培养。
图3A-图3G表明HBs的长期扩增及其鉴定，其中，流式分选纯化hPSC诱导来源的HBs(图3A)；分选获取的HBs在扩增培养条件下具有形成克隆的能力(图3B)；长期扩增后的HBs依然维持较高的增殖能力(图3C-图3D；HBs标志性蛋白表达的检测(图3E)；转录组对比分析扩增前后的HBs(图3F-图3G)。
如图3F-图3G所示，扩增的HBs表达一系列HBs的标志蛋白，如AFP，HNF4，EpCAM，E-CAD，SOX9等；同时，流式细胞术分析结果显示Ki-67和EpCAM双阳性率的细胞将近60％，说明HBs具有较强的增殖能力。在此培养条件下HBs可以连续扩增培养超过50代；优化的肝扩增培养基支持HBs长期扩增，并能很好的维持其肝祖特性；长期扩增后的HBs保持较强的增殖能力。
以上结果表明，本申请建立的HBs扩增培养体系和方法，可以选择性的长期扩增hPSC诱导获取的HBs，并维持其稳定的HBs表型和相关功能。
实施例3诱导HBs分化为功能成熟的肝细胞和胆管细胞
HBs在扩增培养条件下生长至90％融合时更换为成熟肝细胞诱导培养基，即基础培养基HepatoZYME-SFM(Gibco)包含10ng/mL OncostatinM(OSM)，0.1μM dexamethasone(DEX；Sigma-Aldrich)和0.5mM NH4Cl(Sigma-Aldrich)，诱导培养7天，HBs可分化为肝样细胞。
图4A-图4G表明扩增后的HBs维持分化为肝细胞和胆管细胞的双潜能，扩增后的HBs可以高效的诱导分化为肝细胞，并表达相应的标志性蛋白ALB，CK18，HNF6，E-CAD和CYP3A4(图4A-图4B)；诱导分化获取的肝细胞具有较强的代谢解毒、合成尿素等成熟肝细胞功能(图4C-图4F)；HBs分化为胆管上皮细胞(图4G)，扩增后的HBs可以高效的分化为肝样细胞和胆管细胞，说明长期扩增不影响其分化能力；诱导分化获得的肝细胞具有较强的代谢解毒、合成尿素等成熟肝细胞功能；
如图4A-图4G所示，这期间HBs逐渐转变为具有方块状的典型肝细胞上皮样形态；免疫荧光鉴定结果显示，诱导获取的肝样细胞表达成熟肝细胞的多个标志蛋白，如ALB，CK18，HNF6，CYP3A4等；进一步肝细胞功能分析结果显示，诱导获取的肝细胞具有分泌白蛋白，分泌尿素，代谢CYP酶代谢底物以及合成糖原等典型的成熟肝细胞功能；
这些结果表明，本申请建立的HBs扩增培养体系和方法可以使HBs长期扩增后不影响其分化能力，扩增后的HBs可以快速高效的诱导分化为具有成熟功能的肝细胞，具有较强的代谢解毒、合成尿素等成熟肝细胞功能。以上的细胞培养及诱导过程需每天更换培养基。
在Matrigel 3D培养条件下，扩增过的和没有扩增过的HBs在胆管分化培养基中都可以分化形成胆管结构，并表达CK7和CK19等胆管细胞标志蛋白。这些结果说明，扩增过的HBs跟没有扩增培养过的HBs一样，都具有向肝细胞和胆管细胞分化的双潜能。
除了特殊注明的部分，以上细胞培养和诱导所用的细胞因子买自PeproTech，小分子化合物买自Selleck公司。
实施例4移植扩增后的HBs修复急性肝损伤模型小鼠肝脏
首先利用DMN处理免疫缺陷NSI小鼠，建立急性肝损伤模型。然后分别将扩增前、扩增后的HBs移植至肝损伤模型小鼠体内，验证两种细胞在体内增殖分化以及修复损伤肝脏的功能。
图5A-图5I表明移植HBs修复肝损伤模型小鼠图，其中，HBs移植实验示意图(图5A)；HBs移植前后的动物存活率分析(图5B)；病理切片分析细胞移植前后肝组织的形态结构(图5C)；肝组织切片分析细胞移植一周后的细胞归巢效率(图5D-图5F)；移植的HBs可以分化为胆管上皮细胞，再生胆管(图5G)；检测移植HBs的小鼠血清中人源白蛋白的分泌(图5H)；肝组织切片分析细胞移植4周后的人源肝细胞(图5I)。
如图5A-图5I的实验结果所示，扩增前后的两种HBs经脾脏移植后，都可以归巢定植于受体肝脏；移植的HBs可以分化为表达人ALB的肝细胞和表达人CK19的胆管上皮细胞，并参与肝脏损伤实质组织的再生修复以及胆管的再生重建，恢复肝功能并提高肝脏损伤模型小鼠的生存率。
这些结果表明，经本申请建立的HBs扩增培养体系和方法长期培养扩增后的HBs，移植到肝损伤模型小鼠体内，与扩增前的HBs一样，都可以归巢定植到损伤肝脏组织中，参与肝实质组织的修复以及胆管的重建，有效恢复损伤肝脏的功能，并拯救肝损伤模型动物；因此，该发明将有助于快速高效的获取大量的具有功能的肝细胞，适宜于临床肝细胞移植应用以及生物人工肝中的肝细胞反应器等使用。
综上所述，提供了一种扩增培养人肝祖细胞的培养基及其应用，所述培养基配方简洁合理，化学成分明确，无血清，各组分相互配合，协同增效，用于体外长期扩增培养HBs并维持其干性，有助于快速高效的获取大量的具有功能的肝细胞，适宜于临床肝细胞移植应用以及生物人工肝中的肝细胞反应器使用，将为每年新增的百万代偿期/失代偿期肝硬化患者及其急性肝中毒患者的生命带来希望，具有广阔的应用前景和巨大的市场价值。
申请人声明，本申请通过上述实施例来说明本申请的详细方法，但本申请并不局限于上述详细方法，即不意味着本申请必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本申请的任何改进，对本申请产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本申请的保护范围和公开范围之内。

Claims (10)

  1. 一种扩增培养人肝祖细胞的培养基，其中，所述培养基包括液体基础培养基、胰岛素-转铁蛋白-亚硒酸钠混合液、细胞因子、糖原合成激酶3β抑制剂、Hedgehog信号通路激活剂和转化生长因子受体β抑制剂。
  2. 根据权利要求1所述的培养基，其中，所述培养基还包括白蛋白；
    优选地，所述液体基础培养基包括RMPI1640细胞培养基、DMEM/F12细胞培养基、MEM细胞培养基、DMEM细胞培养基、IMDM细胞培养基、199细胞培养基或F10细胞培养基中的任意一种或至少两种的组合；
    优选地，所述白蛋白包括人重组白蛋白和/或牛血清白蛋白；
    优选地，所述白蛋白的质量浓度为5-500μg/mL。
  3. 根据权利要求1或2所述的培养基，其中，所述细胞因子包括骨形态发生生长因子、肝细胞生长因子和表皮生长因子；
    优选地，所述骨形态发生生长因子包括BMP2和/或BMP4；
    优选地，所述骨形态发生生长因子的质量浓度为1-50ng/mL；
    优选地，所述肝细胞生长因子的质量浓度为1-100ng/mL；
    优选地，所述表皮生长因子的质量浓度为1-200ng/mL。
  4. 根据权利要求2或3所述的培养基，其中，所述糖原合成激酶3β抑制剂的浓度为10nM-100μM；
    优选地，所述胰岛素-转铁蛋白-亚硒酸钠混合液的体积百分含量为0.1-10％；
    优选地，所述转化生长因子受体β抑制剂的摩尔浓度为50nM-50μM；
    优选地，所述Hedgehog信号通路激活剂的摩尔浓度为10nM-50μM。
  5. 根据权利要求1-4中任一项所述的培养基，其中，所述糖原合成激酶3β抑制剂包括CHIR99021和/或CHIR98014；
    优选地，所述转化生长因子受体β抑制剂包括A8301、SB431542或E-616452中的任意一种或至少两种的组合；
    优选地，所述Hedgehog信号通路激活剂包括SAG和/或Purmorphaminede。
  6. 一种长期体外扩增培养人肝祖细胞的方法，其中，采用权利要求1-5中任一项所述的培养基。
  7. 根据权利要求6所述的方法，其中，所述方法包括如下步骤：
    (1)获取Ep-CAM +/C-kit -的肝祖细胞；
    (2)采用权利要求1-5中任一项所述的培养基对步骤(1)获取的细胞进行体外培养传代；
    优选地，步骤(1)所述肝祖细胞的来源包括人多能干细胞、肝脏干细胞、肝母细胞或卵圆细胞中的任意一种或至少两种的组合；
    优选地，步骤(1)所述获取的方法包括流式细胞术分选。
  8. 根据权利要求6或7所述的方法，其中，具体包括如下步骤：
    (1)流式细胞术分选获取Ep-CAM +/C-kit -的肝祖细胞，所述肝祖细胞的来源包括人多能干细胞、肝脏干细胞、肝母细胞或卵圆细胞中的任意一种或至少两种的组合；
    (2)采用权利要求1-5中任一项所述的培养基对步骤(1)获取的细胞进行体外培养传代。
  9. 一种肝祖细胞，其中，采用权利要求1-5中任一项所述的培养基或权利要求6-8中任一项所述的方法扩增培养得到。
  10. 一种如权利要求9所述的肝祖细胞在制备治疗肝脏疾病的移植细胞、生物人工肝反应器所用的肝细胞、肝脏组织工程或肝脏疾病体外药物筛选中的应用。
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