Medium additive composition for improving metabolic function of reprogrammed hepatocyte-like cells and use thereof

Abstract

The application discloses a culture medium additive composition for improving the metabolic function of reprogrammed hepatocyte-like cells, which is selected as a carbohydrate metabolism activator, an amino acid metabolism activator and / or a lipid metabolism activator. The application has the following advantages: a systematic technical system for the maturation of reprogrammed hepatocyte functions is established. Through joint analysis of transcriptomics and metabolomics, the differences between reprogrammed hepatocytes and primary mature hepatocytes are systematically analyzed, and the core down-regulation nodes and metabolic bottlenecks in key functional pathways such as carbohydrate metabolism, amino acid metabolism, lipid metabolism, energy metabolism and detoxification metabolism are accurately located. On this basis, a targeted chemical small molecule composition for the above metabolic defects is constructed, and the function is enhanced and the metabolism is reshaped after reprogramming, which effectively promotes the transformation of hepatocyte-like cells to mature hepatocyte state, and significantly improves the metabolic activity and functional stability.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a culture medium additive composition for enhancing the metabolic function of reprogrammed hepatocytes and its application. Background Technology

[0002] Currently, hepatocytes obtained through chemical reprogramming or directed differentiation techniques are quite similar to primary hepatocytes in morphological characteristics and expression levels of some liver-specific markers. However, their metabolic functions still exhibit significant immaturity. Studies have shown that, compared to mature primary hepatocytes, reprogrammed hepatocytes generally exhibit downregulation in key liver functional pathways such as carbohydrate metabolism, amino acid metabolism, and lipid metabolism. This metabolic dysfunction not only limits their application value in drug screening, disease modeling, and in vitro toxicity evaluation but also severely restricts their clinical translational potential in hepatocyte transplantation and liver tissue engineering.

[0003] Especially in the treatment of monogenic hereditary liver diseases, the problem of insufficient functional maturity of reprogrammed hepatocytes is even more prominent. Monogenic hereditary liver diseases, such as urea cycle disorders, familial hypercholesterolemia, α1-antitrypsin deficiency, Wilson's disease, and Crigler-Najjar syndrome, are often caused by gene defects in specific metabolic enzymes, transport proteins, or regulatory factors, leading to overall disruption of the liver's metabolic network. Therefore, functional hepatocytes used to treat these diseases not only need to express the corresponding pathogenic genes but also need to possess mature and complete metabolic pathway activity to restore normal metabolic homeostasis. However, existing reprogrammed hepatocytes generally suffer from insufficient expression of key metabolic enzymes, making it difficult to effectively rebuild normal liver metabolic function and thus limiting their therapeutic effects on hereditary metabolic disorders. Therefore, how to effectively improve the metabolic functional maturity of reprogrammed hepatocytes is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0004] In view of the above-mentioned technical limitations, this application proposes a culture medium additive composition for enhancing the metabolic function of reprogrammed hepatocytes and its application; it overcomes the deficiencies and defects mentioned in the background art.

[0005] To achieve the above objectives, this application adopts the following technical solution: The inventive point of this application is to provide a culture medium additive composition for enhancing the metabolic function of reprogrammed hepatocytes, wherein the additive composition is selected as a carbohydrate metabolism activator, an amino acid metabolism activator, and / or a lipid metabolism activator.

[0006] Optionally, in the above-mentioned culture medium additive composition, the carbohydrate metabolism activator is selected from one or more of trans-2,4-dimethoxystilbene, C24, A-769662, MD001, Ampkinone, oat o-aminobenzamide, scutellarin, sulforaphane, baicalin, BCH, and ganoderic acid T; preferably, it is trans-2,4-dimethoxystilbene at a concentration of 5-15 μmol / L and oat o-aminobenzamide at a concentration of 80-120 μmol / L.

[0007] Optionally, in the above-mentioned culture medium additive composition, the amino acid metabolism activator is selected from one or more of L-ornithine-L-aspartic acid, S-adenosyl-L-methionine, alanyl-glutamine, and a mixture of branched-chain amino acids; preferably, 180-220 μmol / L of S-adenosyl-L-methionine and 3-5 mmol / L of alanyl-glutamine.

[0008] Optionally, in the above-mentioned culture medium additive composition, the lipid metabolism activator is selected from one or more of PPAR agonists, AMPK activators, and FXR agonists; preferably, it is 4-6 μmol / L of PPAR agonist LDT409 and 0.1-0.3 μmol / L of AMPK activator PF-06409577.

[0009] Optionally, in the above-mentioned culture medium additive composition, the PPAR agonist is selected from one or more of LDT409, WY-14643, GW7647, Saroglitazar, MD001, and dihydrosterelic acid; the AMPK activator is selected from one or more of PF-06409577, WS070117, A-769662, and berberine; and the FXR agonist is selected from one or more of Fexaramine, obeticholic acid, LH10, ​​hesperidin, fargesone A, HEC96719, BMS-986318, BAR502, and T0901317.

[0010] The working concentration of the aforementioned small chemical molecules is 0.1 nM to 50 mM, preferably 1 nM to 10 mM, more preferably 10 nM to 5 mM; for ultra-high efficiency agonists, the working concentration can be 0.01 pM to 100 nM.

[0011] The second inventive point of this application is to provide a method for inducing and preparing reprogrammed hepatocytes with enhanced metabolic function, which involves culturing the reprogrammed hepatocytes in vitro with an effective amount of the above-mentioned culture medium additive composition.

[0012] Optionally, in the above-described induction preparation method, the reprogrammed hepatocytes are obtained by sequentially inducing somatic cells with a chemical reprogramming medium and a medium containing small molecule hepatocytes; the culture time after in vitro contact is 24 hours to 14 days; preferably 48 hours to 7 days.

[0013] The third inventive point of this application provides a metabolically enhanced reprogrammed hepatocyte-like cell, which is prepared by the above-described induction preparation method.

[0014] The fourth inventive point of this application provides the use of the above-mentioned culture medium additive composition or the above-mentioned metabolically enhanced reprogrammed hepatocytes in the preparation of biological products for the treatment of monogenic hereditary liver diseases, or in the preparation of cell models for drug screening, toxicological evaluation or gene therapy function verification.

[0015] Optionally, in the above applications, the single-gene hereditary liver disease is selected as phenylketonuria, urea cycle disorder, familial hypercholesterolemia, α1-antitrypsin deficiency, Wilson's disease, or Crigler-Najjar syndrome.

[0016] Current research on the functional immaturity of reprogrammed hepatocytes generally lacks a systematic understanding and mechanistic analysis of metabolic defects, particularly a lack of precise diagnostic strategies based on multi-omics integrated analysis. Existing studies have primarily focused on the application of small molecule compounds in the cell reprogramming induction stage to promote cell fate transitions or as standalone drugs for the treatment of related diseases, with limited interventions targeting functional repair and metabolic maturation of "already reprogrammed hepatocyte-like cells." Therefore, how to further enhance the core metabolic functions of these cells after acquisition remains a key unsolved technical challenge in this field.

[0017] Compared with the prior art, this application has the following advantages: This application establishes a systematic technical framework for the functional maturation of reprogrammed hepatocytes. Through combined transcriptomics and metabolomics analysis, the differences between reprogrammed hepatocytes and primary mature hepatocytes are systematically analyzed, precisely locating key down-regulatory points and metabolic bottlenecks in critical functional pathways such as carbohydrate metabolism, amino acid metabolism, lipid metabolism, energy metabolism, and detoxification metabolism. Based on this, this application further constructs targeted small molecule chemical compositions for the aforementioned metabolic defects. By implementing functional enhancement and metabolic remodeling after reprogramming, it effectively promotes the transformation of hepatocyte-like cells into mature hepatocytes, significantly improving their metabolic activity and functional stability. Attached Figure Description

[0018] Figure 1The image shown is a graph illustrating the detection results of significantly increased levels of the i-CiHep carbohydrate metabolism-related genes Glut2 and Gck, as described in one embodiment of this application.

[0019] Figure 2 The image shown is a graph illustrating the detection results of significantly increased levels of the i-CiHep amino acid metabolism-related genes Cps1 and Otc, as described in one embodiment of this application.

[0020] Figure 3 The image shown is a detection result of significantly increased levels of the i-CiHep lipid metabolism-related genes Cpt1a and Cyp7a1, as described in one embodiment of this application.

[0021] Figure 4 As shown in one embodiment of this application, i-CiHep transplanted into the spleen can effectively correct metabolic defects in phenylketonuria model mice (significantly decreased phenylalanine levels and significantly increased tyrosine levels).

[0022] Figure 5 As shown in one embodiment of this application, i-CiHep transplanted into the spleen can effectively correct metabolic defects in low-density lipoprotein receptor-deficient model mice (significantly reducing serum high-density lipoprotein cholesterol HDL-C and low-density lipoprotein cholesterol LDL-C levels). Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0025] The sources of the formulations used in this application are shown in Table 1 below: Table 1

[0026] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0027] Example 1 Hepatocyte-like cells were induced by somatic cell reprogramming: (1) Preparation of chemical reprogramming medium: Culture skin fibroblasts (MSF) and add small chemical molecules to them: 20 μmol / L CHIR99021, 10 μmol / L 616452, 50 μmol / L Forskolin, 0.05 μmol / LAM580, 5 μmol / L EPZ004777; (2) On the second day after MSF plating, once the cells were stable, the MSF were induced with chemical reprogramming medium and placed in a cell culture incubator at 37°C and 5% CO2 concentration for static culture. (3) Preparation of hepatocyte culture medium containing small chemical molecules: First, prepare hepatocyte culture medium, then add 20 μmol / L CHIR99021, 10 μmol / L 616452 and 50 μmol / L Forskolin. After inducing the cells in MSF with chemical reprogramming medium for 4 days, switch to hepatocyte culture medium containing small chemical molecules for further induction culture. The induction culture is still placed in a cell culture incubator at 37℃ and 5% CO2 concentration for static culture. (4) After induction for 4 days, reduce the content of small chemical molecules in the culture medium in step (3): add only 3 μmol / L CHIR99021, 2 μmol / L 616452, 2 μmol / L Forskolin, and 50 μg / ml Vitamin C for subsequent induction; change the medium once every 4 days during the induction process, and continue induction for 24 to 31 days to obtain somatic cell reprogrammed hepatocytes CiHep.

[0028] The MSF medium is prepared by adding 10% fetal bovine serum, 1% penicillin / streptomycin, and 5 μg / mL bFGF (basic fibroblast growth factor) to DMEM / F12 medium.

[0029] The hepatocyte culture medium was prepared as follows: DMEM / F12 medium was supplemented with 10% FBS, 1% glutamine, 1% sodium pyruvate, 1% penicillin-streptomycin solution, 1% insulin-transferrin-selenium additive, 1 μmol / L dexamethasone, 10 ng / mL EGF, 20 ng / mL HGF, and 20 ng / mL TGF-α.

[0030] Example 2 Experiments and results on improving the carbohydrate metabolism pathway in reprogrammed hepatocytes: Reprogrammed hepatocytes were seeded in a reprogrammed hepatocyte culture medium containing 10 μmol / L trans-2,4-dimethoxystilbene (TDMS) and 100 μmol / L oat oat aminobenzamide (AVNs) and cultured continuously at 37°C and 5% CO2 for 72 hours; the results are as follows. Figure 1 As shown, compared with the untreated control group (i.e., the CiHep hepatocytes prepared in Example 1), the expression of key genes related to glucose uptake / utilization, such as Glut2 and Gck, was upregulated in the small molecule treatment group, consistent with the increased cellular glucose consumption, suggesting that regenerated hepatocytes have enhanced glucose uptake and metabolism capabilities. The upregulation of key genes such as Glut2 and Gck led to a significant increase in cellular glucose consumption, indicating that their carbohydrate metabolism capacity was improved (doi: 10.1007 / s11033-023-08535-w; doi: 10.3389 / fphys.2019.00148).

[0031] The preparation method of the reprogrammed hepatocyte culture medium is as follows: DMEM / F12 culture medium is supplemented with 10% FBS, 1% glutamine, 1% sodium pyruvate, 1% penicillin-streptomycin solution, 1% insulin-transferrin-selenium additive, 1 μmol / L dexamethasone, 10 ng / mL EGF, 20 ng / mL HGF, 20 ng / mL TGF-α, 3 μmol / L CHIR99021, 2 μmol / L 616452, 2 μmol / L Forskolin, and 50 μg / ml Vitamin C.

[0032] Example 3 Experiments and effects on improving the amino acid metabolism pathways of reprogrammed hepatocytes: Reprogrammed hepatocytes were seeded in a culture medium containing 200 μmol / L S-adenosyl-L-methionine and 4 mmol / L alanyl-glutamine, and cultured continuously for 72 hours at 37°C and 5% CO2; the results are as follows. Figure 2 As shown, compared with the untreated control group, the gene expression levels of Cps1 and Otc, key enzymes in the urea cycle, were significantly upregulated in the small molecule treatment group. Since the urea cycle is the core pathway for the liver to process ammonia produced from amino acid breakdown, the upregulation of Cps1 and Otc suggests that regenerated hepatocytes have stronger ammonia clearance and urea synthesis potential, indicating that their nitrogen metabolism function related to amino acid breakdown is enhanced (doi: 10.1146 / annurev.nutr.22.110801.140547.; doi: 10.3389 / fphys.2021.748249.).

[0033] The method for preparing reprogrammed hepatocytes is shown in Example 2.

[0034] Example 4 Experiments and results on improving reprogrammed lipid metabolism pathways in hepatocytes: Reprogrammed hepatocytes were seeded in a culture medium containing 5 μmol / L PPAR α / γ / δ partial agonist LDT409 and 0.2 μmol / L AMPK activator PF-06409577, and cultured continuously at 37℃ and 5% CO2 for 72 hours; the results are as follows. Figure 3 As shown, compared with the untreated control group, the expression levels of lipid metabolism-related genes Cpt1a and Cyp7a1 were significantly upregulated in the small molecule treatment group.

[0035] The method for preparing reprogrammed hepatocytes is shown in Example 2.

[0036] Example 5 Metabolic enhancement reprogrammed hepatocytes transplanted into the spleen effectively correct metabolic defects in a mouse model of monogenic hereditary liver disease. The metabolically enhanced reprogrammed hepatocytes prepared in Example 3 above were implanted into the spleen / liver of phenylketonuria model mice. Figure 4 The results showed that transplanted hepatocytes could correct metabolic defects, and the serum phenylalanine (PAH) level in phenylketonuria model mice was significantly reduced (the reduction was more pronounced in the spleen transplantation group). Continuous dynamic monitoring over 6 months showed that the serum tyrosine (Tyr) levels in both the spleen transplantation group and the liver transplantation group were significantly higher than those in the untreated group. Figure 4 As shown in A), serum phenylalanine (Phe) levels decreased significantly (as shown in A). Figure 4 (As shown in B); among them, the splenic transplantation group showed a greater increase in tyrosine and a more significant decrease in phenylalanine, and the overall metabolic correction effect was better than that of the intrahepatic transplantation group.

[0037] Specific procedures for transplanting metabolically enhanced reprogrammed hepatocytes (i-CiHep) into the spleen: (1) Seven days after the spleen transfer surgery, mice were anesthetized with 1.25% aphthol. After the mice were deeply anesthetized, the hair in the spleen position was cut off with curved scissors and the skin in that area was moistened with 70% ethanol. (2) Place the mouse on its side with its left side facing up on the heating blanket; (3) Metabolically enhanced reprogrammed hepatocytes (5 × 10⁻⁶) were injected intraspleurally using a 31G syringe. 6 (each mouse) (4) About 1 minute after the injection is completed, slowly withdraw the needle and press the injection site with a sterile cotton ball for 3 minutes; (5) Clean the mouse skin with 70% ethanol until the anesthesia wears off, and then put the mouse back into a clean cage; (6) 24 hours after transplantation, the serum of mice was collected and the content of phenylalanine and tyrosine in the serum was detected at different time points (phenylalanine colorimetric kit and tyrosine colorimetric kit).

[0038] The procedure for transplanting metabolically enhanced reprogrammed hepatocytes i-CiHep into the liver is the same as described above. The enhanced hepatocytes obtained in Example 3 are transplanted into the spleen of an unmodified phenylketonuria model mouse. The cells are then able to migrate to the liver and be localized there to perform their function.

[0039] Example 6 Serum phenylalanine content detection: (1) Detection principle: Phenylalanine (Phe), chemically known as 2-amino-3-phenylpropionic acid, is an essential amino acid that participates in various biosynthetic processes. In the body, most phenylalanine is oxidized to tyrosine by phenylalanine hydroxylase, and together with tyrosine, it synthesizes important neurotransmitters and hormones, participating in the body's carbohydrate and lipid metabolism. Phe can react with a chromogenic agent under the action of enzymes to produce a chromogenic substance with a maximum absorption peak at 450 nm. The Phe content of the sample can be calculated by measuring the OD value of this chromogenic substance at 450 nm.

[0040] (2) Reagents and supplies: As shown in Table 2.

[0041] Table 2

[0042] (3) Sample processing: Centrifuge at 12000 g for 15 min at 4°C using a 10 KD ultrafiltration tube, collect the filtrate, add 1 μL of reagent II to every 200 μL of filtrate sample, let stand at 25°C for 10 min, and place on ice for testing.

[0043] (4) Operating steps: ① Standard wells: Add 20 μL of standard solutions of different concentrations to the corresponding enzyme-labeled wells.

[0044] Measurement wells: Take 20 μL of the sample to be tested and add it to the corresponding enzyme label well.

[0045] Control wells: Take 20 μL of the sample to be tested and add it to the corresponding enzyme-labeled well.

[0046] ② Add 20 μL of reagent 5 to each well in step ①.

[0047] ③ Add 100 μL of the working solution to the standard well and the measurement well in step ②.

[0048] ④ Add 100 μL of control working solution to the control well in step ②.

[0049] ⑤ Shake the plate for 5 seconds, incubate at 37°C for 30 minutes, and use a microplate reader to detect the OD value of each well at 450 nm.

[0050] Example 7 Serum tyrosine content detection: (1) Detection principle: This detection is based on the enzymatic oxidation of tyrosine to generate a stable signal (OD 492 nm), which is proportional to the tyrosine content.

[0051] (2) Kit components: Assay Buffer V / Tyr Assay Buffer (Detection Buffer V / Tyrosine Detection Buffer); Tyrosine Enzyme Mix / Tyr Enzyme Mix (Tyrosinase Mix); Tyrosine / Tyr Standard.

[0052] (3) Sample preparation: Add the sample to a 10 kDa ultrafiltration column and centrifuge at 10,000 × g for 10 minutes at 4°C. Collect the filtrate. Add 80–135 μL of filtrate to the required wells in a 96-well plate. Make up the volume of each well to 150 μL with dH2O.

[0053] (4) Preparation of standard curve: ① Dilute Tyrosine / Tyrosine Standard to 2.5 mM: Take 25 μL of 100 mM Tyrosine / Tyrosine Standard and add it to 975 μL of dH2O.

[0054] ② Add 0, 2, 6, 12, 18, 24 and 30 μL of Tyrosine / Tyrosine Standard to a series of wells in a 96-well plate to prepare standard curves containing 0, 5, 15, 30, 45, 60 and 75 nmol of tyrosine standard per well.

[0055] ③ Make up the volume of each well to 150 μL using Assay Buffer V / Tyr Assay Buffer. The diluted standard can be stored at 4°C for subsequent testing.

[0056] (5) Reaction mixture: Prepare sufficient reaction mixture according to the number of wells to be tested. Prepare 50 μL of mixture per well, as shown in Table 3: Table 3

[0057] Add 50 μL of reaction mixture to each well containing the standard and the sample.

[0058] (6) Place the well plate at room temperature and incubate in the dark for 60 minutes.

[0059] (7) Use an ELISA reader to measure absorbance (OD 492 nm).

[0060] Example 8: Animal experiments demonstrating the enhancement of lipid metabolism pathways were conducted as follows: The transplantation method for lipid metabolism-enhanced hepatocytes was similar to that in Example 5, and the animal model selected was a familial hypercholesterolemia mouse model (low-density lipoprotein receptor-deficient mouse model). Experimental results are as follows: Figure 5 As shown, by detecting serum lipid metabolism-related indicators, the therapeutic effect and metabolic improvement of familial hypercholesterolemia were systematically evaluated. Figure 5 The results showed that, compared with intrahepatic transplantation, intrasplenic transplantation significantly reduced serum high-density lipoprotein cholesterol (HDL-C). Figure 5 A) and low-density lipoprotein cholesterol (LDL-C, Figure 5 B) levels, and improves high-density lipoprotein cholesterol levels, with overall treatment effects significantly superior to intrahepatic transplantation.

[0061] Serum levels of HDL-C and LDL-C were measured 24 hours after cell transplantation. HDL-C was measured using the HDL-C colorimetric assay kit (Elabscience, catalog number: E-BC-K221-M), and LDL-C was measured using the tyrosine LDL-colorimetric assay kit (Elabscience, catalog number: E-BC-K205-M).

[0062] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A culture medium additive composition for enhancing the metabolic function of reprogrammed hepatocyte-like cells, characterized in that, The additive composition is selected as a carbohydrate metabolism activator, an amino acid metabolism activator, and / or a lipid metabolism activator.

2. The culture medium additive composition according to claim 1, characterized in that, The carbohydrate metabolism activator is selected from one or more of trans-2,4-dimethoxystilbene, C24, A-769662, MD001, Ampkinone, oat o-aminobenzamide, scutellarin, sulforaphane, baicalin, BCH, and ganoderic acid T; preferably, it is trans-2,4-dimethoxystilbene at a concentration of 5-15 μmol / L and oat o-aminobenzamide at a concentration of 80-120 μmol / L.

3. The culture medium additive composition according to claim 1, characterized in that, The amino acid metabolism activator is selected from one or more of L-ornithine-L-aspartic acid, S-adenosyl-L-methionine, alanyl-glutamine, and a mixture of branched-chain amino acids; preferably, it is 180-220 μmol / L of S-adenosyl-L-methionine and 3-5 mmol / L of alanyl-glutamine.

4. The culture medium additive composition according to claim 1, characterized in that, The lipid metabolism activator is selected from one or more of PPAR agonists, AMPK activators, and FXR agonists; preferably, it is the PPAR agonist LDT409 at 4-6 μmol / L and the AMPK activator PF-06409577 at 0.1-0.3 μmol / L.

5. The culture medium additive composition according to claim 4, characterized in that, The PPAR agonist is selected from one or more of LDT409, WY-14643, GW7647, Saroglitazar, MD001, and dihydrosterol; the AMPK activator is selected from one or more of PF-06409577, WS070117, A-769662, and berberine; and the FXR agonist is selected from one or more of Fexaramine, obeticholic acid, LH10, ​​hesperidin, fargesone A, HEC96719, BMS-986318, BAR502, and T0901317.

6. A method for inducing and preparing reprogrammed hepatocytes with enhanced metabolic function, characterized in that, The reprogrammed hepatocytes are cultured in vitro after being contacted with an effective amount of the culture medium additive composition according to any one of claims 1-5.

7. The induced preparation method according to claim 6, characterized in that, The reprogrammed hepatocytes were obtained by sequentially inducing somatic cells with a chemical reprogramming medium and a medium containing small molecule hepatocytes; the culture time after in vitro contact was 24 hours to 14 days.

8. A metabolically enhanced reprogrammed hepatocyte-like cell, characterized in that, The hepatocytes were prepared by the induction preparation method described in claim 6 or 7.

9. The culture medium additive composition according to any one of claims 1-5 or the metabolically enhanced reprogrammed hepatocytes according to claim 8, in the preparation of biological products for the treatment of monogenic hereditary liver diseases, or in the preparation of cell models for drug screening, toxicological evaluation or gene therapy functional verification.

10. The application according to claim 9, characterized in that, The selected single-gene hereditary liver diseases are phenylketonuria, urea cycle disorder, familial hypercholesterolemia, α1-antitrypsin deficiency, Wilson's disease, or Crigler-Najjar syndrome.

Images