Patent ID: 12234482

DETAILED DESCRIPTION

To further elaborate on the technical means adopted and the effects achieved in the present application, the solutions of the present application are further described below through specific examples in conjunction with drawings, but the present application is not limited to the scope of the examples.

Example 1 Preparation of a Medium

A basal medium, i.e., RPMI1640 was supplemented with 50 μg/mL of human recombinant albumin and 1% (volume percent content) of an insulin-transferrin-sodium selenite (ITS) mixture.

HBs expansion culture media were prepared, including the above basal medium and an expansion combination: B20CEH, AB20CEH, AB10CEHS, ACEHS, or ACEH. Letters in the expansion combination have the following meanings: A represents 50 nM to 50 μM of transforming growth factor β (TGF-β) receptor inhibitor A8301; B10or B20represents 10 ng/mL or 10 ng/mL of a bone morphogenetic growth factor (BMP2 or BMP4), respectively; C represents 10 nM to 10 μM of a glycogen synthase kinase 3β (GSK3β) inhibitor; E represents 1 ng/mL to 200 ng/mL of epidermal growth factor (EGF); H represents 1 ng/mL to 100 ng/mL of hepatocyte growth factor (HGF), and S represents 10 nM to 50 μM of a Hedgehog signaling pathway activator.

An optimized HBs expansion culture medium was prepared, i.e. including the above basal medium and the expansion combination AB10CEHS, i.e. including 5 μM of A8301, 20 ng/mL of BMP4, 3 μM of CHIR99021, 20 ng/mL of EGF, 20 ng/mL of HGF, and 0.5 μM of SAG. Cells were digested with Accutase enzyme and subcultured when grown to 80% confluency.

Example 2 Expansion of HBs Obtained Through hPSC Differentiation

hPSCs (H1 or iPSCs from the Key Laboratory of Stem Cells) were cultured in a 12-well plate coated with 1% Matrigel (a growth factor reduced, BD Bioscience) in culture medium mTeSR1 (Stem Cell Technologies). The culture medium was changed to a culture medium for DE-directed induction when the hPSCs grew at 60%-70% confluency, where RPMI1640 contained 2% of B27 (minus insulin, Invitrogen), 3 μM of CHIR99021 (CHIR) and 100 ng/mL of Activin A. The hPSCs were induced for one day and then induced for another two days with CHIR removed. At this time, the cells differentiated into DE cells. Then, the culture medium was changed to a culture medium for directed differentiation into hepatoblasts, where RPMI1640 contained 2% of B27 (Invitrogen), 20 ng/mL BMP2, 20 ng/mL of BMP4 and 30 ng/mL of FGF4, and the DE cells were induced for four days. The culture medium was then replaced with RPMI1640 that contained 2% of B27, 20 ng/mL of BMP4 and 20 ng/mL of HGF4 and the DE cells were induced for another three says and further differentiated into HBs.

An induction process of HBs and the identification of cells at each relevant stage are shown inFIGS.1A to1D, wherein: a flowchart showing directed differentiation of hPSCs to liver lineage (FIG.1A), changes in cell morphology in the induction process of HBs (FIG.1B), immunofluorescence for identifying DE cells on Day 3 and HBs on Day 10 (FIG.1C), and flow cytometry for analyzing the expression of marker proteins and the expression of cell proliferation marker protein Ki67 at different stages of differentiation into HBs (FIG.1D).

hPSCs may be induced to differentiate into HBs which have a strong capability to proliferate. HBs may be expanded in large amounts under suitable culture conditions, and may further rapidly differentiate into functional mature hepatocytes and cholangiocytes. Therefore, large-scale expansion of HBs obtained through inducing hPSCs provides a source for rapid and efficient acquisition of a large number of functional hepatocytes, and thus is an effective method for meeting the requirement for a large number of hepatocytes in clinical hepatocyte transplantation and hepatocyte reactors in bioartificial liver.

At present, an effective method for long-term culturing and expanding HBs in vitro has not been established in the industry. Difficulties lie in the lack of a deep understanding of the process and regulation mechanism of the differentiation of hPSCs into liver cells and the unclear regulation mechanisms of HB proliferation and sternness maintenance. Therefore, the regulation mechanism during directed differentiation of hPSCs to liver lineage is to be elucidated, the regulation mechanism during HB proliferation and stemness maintenance is to be clarified, and a method for expanding and culturing HBs whose formula has clear chemical components and is serum-free is to be established so that HBs can be expanded and cultured to a large scale, which will facilitate the rapid and efficient acquisition of a large number of functional hepatocytes and the application of HBs to the treatment of liver diseases such as cell transplantation and bioartificial liver dialysis.

Based on an established method for inducing directed differentiation to liver lineage, signaling pathways related to proliferation and differentiation of HBs are analyzed, the regulation mechanisms of key signaling pathways such as Wnt, TGF-β, BMP, and Hedgehog on HB proliferation and maintenance of liver progenitor characteristics are clarified, and cytokines and small molecule compounds are further adopted to regulate related signaling pathways, so as to develop a formula of a culture medium which supports stable proliferation of HBs and better maintains the liver progenitor characteristics.

First, during differentiation of hPSCs into HBs, the activity of the signaling pathways related to the proliferation and differentiation of HBs was quantitatively analyzed through RT-PCR. The results showed that the activity of the signaling pathways such as Wnt or Hedgehog related to the proliferation of HBs was down-regulated during the differentiation (FIG.2A).

Further, effects of the signaling pathways on the proliferation and differentiation of HBs were analyzed by regulating the related signaling pathways with small molecules. The results showed that CHIR, A8301 and SAG promoted the proliferation of HBs (FIGS.2B and2C).

In addition to proliferation, the effects of the related signaling pathways on the characteristics of HBs were further analyzed. The results showed that CHIR promoted the proliferation of HBs and inhibited the expression of AFP, that is, inhibited the maintenance of the characteristics of HBs. Whereas, A8301 and SAG promoted the expression of liver cell-related genes such as AFP, that is, promoted the maintenance of the characteristics of HBs (FIG.2D).

The above results showed the balance of the regulation of the related signaling pathways such as Wnt, TGF-β, BMP, and Hedgehog on the proliferation and differentiation of HBs (FIG.2E).

Based on this, small molecule compounds were further screened to optimize the formula for the expansion and culture of HBs (FIG.2F). It was found through expansion and culture that multiple formulas of the culture medium promoted the proliferation of HBs (FIGS.2G and2H). However, different formulas of the culture medium had different effects in maintaining the characteristics of HBs. The optimized formula AB10CEHS of the culture medium promoted the proliferation of HBs while maintaining the liver progenitor characteristics (FIGS.2I and2J). The effects of different formulas of the culture medium on the maintenance of the characteristics of HBs are shown in Table 1. Letters in the expansion combination have the following meanings: A represents 50 nM to 50 μM of transforming growth factor β (TGF-β) receptor inhibitor A8301; B10or B20represents 10 ng/mL or 10 ng/mL of a bone morphogenetic growth factor (BMP2 or BMP4), respectively; C represents 10 nM to 10 μM of a glycogen synthase kinase 33 (GSK3β) inhibitor; E represents 1 ng/mL to 200 ng/mL of epidermal growth factor (EGF); H represents 1 ng/mL to 100 ng/mL of hepatocyte growth factor (HGF), and S represents 10 nM to 50 μM of a Hedgehog signaling pathway activator.

TABLE 1TreatmentAFP+HNF4α+B20CEH81.8% +/− 4.283.4% +/− 4.8AB20CEH94.5% +/− 3.396.2% +/− 2.3AB10CEHS96.7% +/− 5.498.8% +/− 6.0ACEHS77.2% +/− 5.086.2% +/− 4.5ACEH72.7% +/− 5.281.5% +/− 6.5

The HBs obtained through the differentiation of hPSCs were digested into single cells by Accutase (life), resuspended in PBS containing 3% of BSA, and further incubated with Ep-CAM (Milteny) and C-Kit (BD Biosciences) antibodies. Then, Ep-CAM+/C-Kit−HBs were sorted through flow cytometry. The sorted HBs were expanded and cultured in a plate coated with 1% Matrigel in optimized HB expansion medium AB10CEHS, that is, basal medium RPMI1640 containing 2% of B27 and 1% of ITS (life), containing cytokines: 20 ng/mL of BMP4 and 20 ng/mL of EGF, and small molecule compounds: 20 ng/mL of HGF, 3 μM of CHIR, 5 μM of A8301, and 0.5 μM of SAG. Cells were digested with Accutase and subcultured when grown to 80% confluency.

FIGS.3A to3Gshow the long-term expansion and identification of HBs, wherein: a diagram showing purification of HBs induced from hPSCs through flow cytometry sorting (FIG.3A), a diagram illustrating the capability of the sorted HBs to form clones under expansion and culture conditions (FIG.3B), diagrams illustrating that HBs expanded for a long term still maintained a relatively high capability to proliferate (FIGS.3C and3D), a diagram showing detected expression of marker proteins of HBs (FIG.3E), and diagrams showing analysis of HBs before and after expansion through a comparison of transcriptome (FIGS.3F and3G).

As shown inFIGS.3F and3G, the expanded HBs expressed a series of marker proteins of HBs, such as AFP, HNF4, EpCAM, E-CAD, and SOX9. Meanwhile, the results of flow cytometry showed that nearly 60% of cells were positive for both Ki-67 and EpCAM, which indicates that HBs have a strong capability to proliferate. Under these culture conditions, HBs can be continuously expanded and cultured for more than 50 passages. The optimized liver expansion medium allows HBs to be expanded for a long term while maintaining their liver progenitor characteristics. The HBs expanded for a long term maintain a strong capability to proliferate.

The above results show that the system and method for expanding and culturing HBs, which are established in the present application, can selectively expand HBs obtained by inducing hPSCs for a long time while maintaining stable HB phenotypes and related functions.

Example 3 HBs Being Induced to Differentiate into Functional Mature Hepatocytes and Cholangiocytes

HBs were grown to 90% confluency under expansion and culture conditions, then the culture medium was changed to a mature hepatocyte induction medium, that is, basal medium HepatoZYME-SFM (Gibco) containing 10 ng/mL of OncostatinM (OSM), 0.1 μM of dexamethasone (DEX; Sigma-Aldrich) and 0.5 mM of NH4Cl (Sigma-Aldrich). The HBs were induced for seven days and differentiated into hepatocyte-like cells.

FIGS.4A to4Gshow that the expanded HBs maintained the dual potential to differentiate into hepatocytes and cholangiocytes. The expanded HBs were efficiently induced to differentiate into hepatocytes and expressed corresponding marker proteins ALB, CK18, HNF6, E-CAD, and CYP3A4 (FIGS.4A and4B). The hepatocytes obtained through induction of differentiation had strong functions of mature hepatocytes, such as metabolic detoxification and urea synthesis (FIGS.4C and4F). HBs were differentiated into bile duct epithelial cells (FIG.4G). Effective differentiation of the expanded HBs into hepatocyte-like cells and cholangiocytes indicates that the long-term expansion does not affect the capability of HBs to differentiate. The hepatocytes obtained through induction of differentiation had strong functions of mature hepatocytes, such as metabolic detoxification and urea synthesis.

As shown inFIGS.4A to4G, HBs gradually transformed into square typical hepatocyte epithelioid morphology. Immunofluorescence results showed that the hepatocyte-like cells obtained through induction expressed multiple marker proteins of mature hepatocytes, such as ALB, CK18, HNF6, and CYP3A4. A further analysis of hepatocyte functions showed that the hepatocytes obtained through induction had typical functions of mature hepatocytes, such as albumin secretion, urea secretion, metabolism of substrates of CYP enzyme and glycogen synthesis.

These results show that the system and method for expanding and culturing HBs, which are established in the present application, can expand HBs for a long term without affecting their capability to differentiate, and the expanded HBs may be rapidly and efficiently induced to differentiate into mature hepatocytes with strong function of mature hepatocytes, such as metabolic detoxification and urea synthesis. The culture medium needs to be changed every day in the above cell culture and induction process.

Under Matrigel 3D culture conditions, both expanded and unexpanded HBs were differentiated into bile duct structures in a bile duct differentiation medium and expressed cholangiocyte marker proteins such as CK7 and CK19. These results suggest that the expanded HBs have the same dual potential to differentiate into hepatocytes and cholangiocytes as the unexpanded HBs. Unless specially noted, the cytokines used for cell culture and induction were purchased from PeproTech and the small molecule compounds were purchased from Selleck.

Example 4 Transplantation of Expanded HBs to Repair the Liver in Mouse Models of Acute Liver Injury

Immunodeficient NSI mice were treated with DMN to establish an acute liver injury model. HBs before and after expansion were transplanted into the mouse models of liver injury to verify the proliferation and differentiation of the HBs before and after expansion as well as functions thereof to repair injured liver in vivo.

FIGS.5A to5Ishow diagrams of the transplantation of HBs to repair mouse models of liver injury, wherein: a schematic diagram illustrating an HB transplantation experiment (FIG.5A), analysis of animal survival rates before and after HB transplantation (FIG.5B), pathology sections for the analysis of the morphology of liver tissues before and after cell transplantation (FIG.5C), liver tissue sections for the analysis of cell homing efficiency after one week of cell transplantation (FIGS.5D to5F), differentiation of the transplanted HBs into bile duct epithelial cells and regeneration bile ducts (FIG.5G), detection of secretion of human albumin in the serum of mice transplanted with HBs (FIG.5H), and liver tissue sections for the analysis of human-derived hepatocytes after four weeks of cell transplantation (FIG.5I).

As shown by experimental results inFIGS.5A to5I, HBs before and after expansion were both homed and colonized in recipient liver after splenic transplantation; and the transplanted HBs were differentiated into hepatocytes expressing human ALB and bile duct epithelial cells expressing CK19, participated in the regeneration and repair of liver injury parenchyma and the regeneration and reconstruction of bile ducts, restored liver functions, and improved the survival rate of liver injury model mice.

These results show that when transplanted into the liver injury model mice, the HBs expanded and cultured for a long term through the system and method for expanding and culturing HBs, which are established in the present application, can be homed and colonized into injured liver tissues, participate in the repair of liver parenchyma and the reconstruction of bile ducts, effectively recover the function of the injured liver, and save liver injury model animals, just like HBs before expansion. Therefore, the present disclosure will facilitate the rapid and efficient acquisition of a large number of functional hepatocytes and is suitable for use in clinical hepatocyte transplantation and hepatocyte reactors in bioartificial liver.

To conclude, the present application provides a culture medium for expanding and culturing human hepatoblasts and use thereof. The culture medium is simple and reasonable in formula, clear in chemical composition, and serum-free, have components that cooperate with each other for synergy, and is used for expanding and culturing HBs in vitro for a long term and maintaining the stemness of HBs, conducive to the rapid and efficient acquisition of a large number of functional hepatocytes, and suitable for use in clinical hepatocyte transplantation and hepatocyte reactors in bioartificial liver. The present application will bring hope for the lives of millions of new liver cirrhosis patients at a compensation/decompensation stage and patients with acute hepatotoxicity and has a broad application prospect and a huge market value.

The applicant has stated that although the detailed method of the present application is described through the examples described above, the present application is not limited to the detailed method described above, which means that implementation of the present application does not necessarily depend on the detailed method described above. It should be apparent to those skilled in the art that any improvements made to the present application, equivalent replacements of raw materials of the product of the present application, additions of adjuvant ingredients to the product of the present application, and selections of specific manners, etc., all fall within the protection scope and the disclosed scope of the present application.