Here, we present a protocol to generate a human liver chimeric mouse model of familial hypercholesterolemia using human induced pluripotent stem cell-derived hepatocytes. This is a valuable model for testing new therapies for hypercholesterolemia.
Familial hypercholesterolemia (FH) is mostly caused by low-density lipoprotein receptor (LDLR) mutations and results in an increased risk of early-onset cardiovascular disease due to marked elevation of LDL cholesterol (LDL-C) in blood. Statins are the first line of lipid-lowering drugs for treating FH and other types of hypercholesterolemia, but new approaches are emerging, in particular PCSK9 antibodies, which are now being tested in clinical trials. To explore novel therapeutic approaches for FH, either new drugs or new formulations, we need appropriate in vivo models. However, differences in the lipid metabolic profiles compared to humans are a key problem of the available animal models of FH. To address this issue, we have generated a human liver chimeric mouse model using FH induced pluripotent stem cell (iPSC)-derived hepatocytes (iHeps). We used Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mice to avoid immune rejection of transplanted human cells and to assess the effect of LDLR-deficient iHeps in an LDLR null background. Transplanted FH iHeps could repopulate 5-10% of the LRG mouse liver based on human albumin staining. Moreover, the engrafted iHeps responded to lipid-lowering drugs and recapitulated clinical observations of increased efficacy of PCSK9 antibodies compared to statins. Our human liver chimeric model could thus be useful for preclinical testing of new therapies to FH. Using the same protocol, similar human liver chimeric mice for other FH genetic variants, or mutations corresponding to other inherited liver diseases, may also be generated.
Low-density lipoprotein receptor (LDLR) captures LDL cholesterol (LDL-C) in blood to modulate cholesterol synthesis in the liver. Mutations in the LDLR gene are the most frequent cause of familial hypercholesterolemia (FH)1. Statins have traditionally been the first line of medication to treat FH and other types of hypercholesterolemia (inherited or acquired). Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A reductase to lower cholesterol synthesis in the liver2. Additionally, statins increase LDLR levels on the hepatocyte surface to promote plasma LDL-C clearance. However, a major caveat of treatment with statins is that they simultaneously induce the expression of proprotein convertase subtilisin/hexin 9 (PCSK9), an enzyme that binds to LDLR to promote its degradation3. This effect is responsible for the insufficient or even null response to statins observed in many patients. Studying this mechanism has, unexpectedly, led to the discovery of an alternative way to treat hypercholesterolemia. PCSK9 antibodies recently approved by the FDA are currently being used in clinical trials and show higher efficacy and better tolerance than statins4. The success of PCSK9 antibodies also implies that there may be other therapeutic possibilities to modulate the LDLR degradation pathway (besides PCSK9) in patients with hypercholesterolemia. Similarly, there is interest in developing new inhibitors of PCSK9 other than antibodies, for example, siRNA oligos5.
To test new therapies for FH and in general any other type of hypercholesterolemia, appropriate in vivo models are necessary. A major problem of current in vivo models, mostly mice6 and rabbits7, are their physiological differences with humans. Crucially, these problems include a different lipid metabolic profile. The generation of human liver chimeric animals8 might help overcome this caveat. The human liver chimeric mouse is a type of "humanized" mouse with its liver repopulated with human hepatocytes, for example, primary human hepatocytes (pHH)9. A problem with pHH is that they cannot be expanded ex vivo, quickly lose their function upon isolation, and are a limited source. An alternative to pHH is the use of induced pluripotent stem cells (iPSC)-derived hepatocytes (iHeps)10. Notably, iPSCs are patient-specific and can be grown indefinitely, so iHeps can be produced on demand, which is a significant advantage over fresh pHH. Moreover, iPSCs can also be easily genetically engineered with designer nucleases to correct or introduce mutations in an isogenic background to allow more faithful comparisons11.
Human liver chimeric mouse with engrafted pHH show similarities to humans in liver metabolic profiles, drug responses, and susceptibility to hepatitis virus infection12. This makes them a good model to study hyperlipidemia in vivo. The most widely used mouse models are based on the Fah-/-/Rag2-/-/Il2rg-/- (FRG) mouse13 and the uPA transgenic mouse8, in which up to 95% of the mouse liver can be replaced by pHH. Interestingly, a recent report described a human FH liver chimeric mouse (based on the FRG mouse) with pHH from a patient carrying a homozygous LDLR mutation14. In this model, the repopulated human hepatocytes had no functional LDLR, but the residual mouse hepatocytes did, thus reducing the utility for performing in vivo testing of drugs relying on the LDLR pathway.
Here, we report a detailed protocol based on our recently published work15 for engrafting FH iHeps into the Ldlr-/-/Rag2-/-/Il2rg-/- (LRG) mouse liver. This human liver chimeric mouse is useful for modeling FH and performing drug testing in vivo.
All methods described here that involve the use of animals have been approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong.
1. Mouse Preparation and Phenotypic Testing
2. iHep Differentiation and Dissociation
LDLR heterozygous KO (+/-) or homozygous KO (-/-) human iPSCs, or FH patient-iPSCs with heterozygous mutations in LDLR (FH iPSCs) are used to produce iHeps. The generation of LDLR +/- or -/- iPSCs and FH iPSCs is described in our previous report15.
3. Intrasplenic Injection of iHeps
4. Test of Plasma LDL-C Level
5. In Vivo Drug Testing in Chimeric Mice Engrafted with LDLR +/- and FH iHeps
6. Endothelial Function Test
Endothelial function is affected early in FH and can be tested in our mouse model as an indicator of the severity of the disease or to evaluate the improvement with different treatments. A stereomicrocope, dissection forceps, scissors, a wire myograph, acquisition hardware (see Table of Materials), and a computer are needed for this.
7. Evidence of iHep Repopulation in the Mouse Liver
Directed Differentiation of Human iPSCs into iHeps
When reaching 70% confluence, human iPSCs are differentiated into iHeps with a 3-step protocol16 (Figure 1 upper panel). After 3 days of endoderm differentiation, iPSC colonies become loosened and spread to full confluence (Figure 1 lower panel). Then, with 2nd stage medium, hepatoblasts appear and proliferate. These cells are crowded but show clear edges at this stage (day 7, Figure 1 lower panel). After 17 days of differentiation, polarized iHeps with typical hexagon morphology appear (Figure 1 lower panel). These iHeps express pHH markers, including AAT and ALB (Figure 2A). Moreover, the ratio of ASGPR+ iHeps should be relatively high, as measured by flow cytometry (Figure 2B)15.
Generation of a FH In Vivo Disease Model Using FH iHeps
To help LRG mice develop hypercholesterolemia, we feed them with a HFHC diet 7 days prior to engraftment (day -7). On the day of engraftment (day 0), LRG mice display around 3-fold plasma LDL-C level (around 600 mg/dL, Figure 3B). Day 15–17 iHeps are engrafted into LRG mice via intrasplenic injection; these iHeps soon reside in the liver parenchyma and proliferate there (Figure 3A). At the end-point, livers of these chimeric mice are collected and fixed, then stained with hALB and hNA, both of which should show clear proof of iHep-mediated liver repopulation in LRG mouse liver based on staining for hALB and hNA (Figure 4A-E). In our hands, both LDLR +/- and FH iHeps could reduce plasma LDL-C level significantly 21 days post-engraftment (Figure 4F). At this point, FH human liver chimeric mice can be used to test therapies for FH.
Validation of FH Human Liver Chimeric Mouse Model Using Available Drugs
To validate our model, we used 2 well-known LDL-C lowering drugs, simvastatin and PCSK9 antibodies (Figure 5A). Our data demonstrate that 21 days after treatment, PCSK9 antibodies have a stronger ability for LDL-lowering and EDV than simvastatin in FH chimeric mice (Figure 5B-D). Notably, the observed percentage reduction of plasma LDL-C with PCSK9 antibodies in FH chimeric mice is similar to that reported in clinical trials4. These results demonstrate the potential utility of LRG chimeric mice engrafted with FH iHeps for preclinical testing of novel drugs for FH.
Figure 1: Directed Differentiation of Human iPSCs into iHeps.Top panel, Timeline for iPSC differentiation into iHeps; key cytokines and media are shown for each stage. (Lower panel) Representative phase contrast images of different time points of iHep differentiation. KSR: knockout serum replacement; HCM: hepatocytes culture medium; HGF: hepatocyte growth factor; OSM: oncostatin M. Please click here to view a larger version of this figure.
Figure 2: Characterization of iHeps Produced with Modified Differentiation Protocol. (A) Immunofluorescence of iHeps at various stages of differentiation. Nuclei are stained in blue in the merged compositions. (B) Bar graph shows the percentage of ASGPR+ iHeps derived obtained at day 17, as measured by flow cytometry. Samples were measured in 3 independent experiments; mean values are shown and error bars indicate standard deviation (SD). Please click here to view a larger version of this figure.
Figure 3: Generation of a FH In Vivo Disease Model with iHeps. (A) Time line for the generation of human liver chimeric mice by intrasplenic injection of FH iHeps into LRG mice. (B) Bar graph shows that feeding LRG mice with HFHC diet leads to significantly increased LDL-C level (n = 10). P values are indicated on the figure and were obtained using an unpaired t-test; mean values are shown and error bars indicate standard error of the mean (SEM). Panel B is modified from Figure 3I of our previous report15. Please click here to view a larger version of this figure.
Figure 4: iHep Mediated Repopulation of LRG Mice Liver. (A) Representative whole section-scanned image of hALB staining in a mouse liver repopulated with LDLR +/- iHeps. Arrows indicate clusters of human iHeps engrafted into the mouse liver; zoomed sections are shown in the right panel. (B) Scatter plot graph shows the percentage of repopulated hALB+ corresponding to iHep-containing areas in a mouse liver (from different donor iPSCs), calculation was based on whole section-scanned images (n = 19). Mean values are shown and error bars indicate SD. (C) Representative images of immunohistochemical staining for hNA in a mouse liver with engrafted FH iHeps. (D) Bar graph shows the percentage of repopulated hNA+ iHeps (from different donor iPSCs) in LRG mouse livers (n = 3). Mean values are shown and error bars indicate SD. (E) hALB and hNA staining on two consecutive sections of a mouse liver repopulated with wild type iHeps. (F) Bar graph shows the percentage of plasma LDL-C reduction from baseline at day 21 post-engraftment; n = 5 for LDLR +/- iHeps and n = 6 for FH iHeps and the vehicle. P values are indicated on the figure and were obtained using an unpaired t-test; mean values are shown error bars indicate SEM. Panels B, D, and E are modified from Figure 3G-3I of our previous report15. Please click here to view a larger version of this figure.
Figure 5: PCSK9 Antibodies Show Stronger LDL Lowering Ability than Simvastatin in Human Liver Chimeric LRG Mice. (A) schematic view of the in vivo drug testing approach using FH human liver chimeric mice. (B and C) Percentage change of plasma LDL-C from baseline at days 14, 21, and 28 in FH chimeric mice fed with HFHC diet and treated with the indicated drugs; n indicates number of mice. P values were obtained using a Kruskal-Wallis test; mean values are shown and error bars indicate SEM. (D) EDV in response to increasing concentrations of ACh. P values are indicated on the figure for indicated concentration. P values were obtained using two-way ANOVA adjusted with Dunnett's multiple comparison; error bars indicate SEM. Figure 5 is modified from Figure 4A–4C and Figure 5A of our previous report15. Please click here to view a larger version of this figure.
Previous studies using iHeps in rodents have confirmed that they are an effective way to study inherited liver diseases17. To further expand the use of this technology and because current FH animal models are suboptimal, we engrafted FH iHeps into LRG mice and showed that the engrafted LDLR +/- or heterozygous LDLR-mutated FH iHeps can reduce mice plasma LDL-C level and respond to lipid-lowering drugs in vivo.
There are 3 critical steps in our protocol for generating human FH liver chimeric mice using iHeps:
1) Production of high-quality iHeps through directed differentiation. Given the clonal variability between iPSC lines18, it is important to use isogenic iPSCs for proper comparisons and to test the iHep differentiation efficiency of the mother cell line before performing the engineering and the engraftment.
2) Correct iHep upload into the syringe. Differently from other protocols, we use 50% extracellular matrix (final concentration, v/v) to resuspend iHeps, and then upload them into the insulin syringe. We believe that the extracellular matrix protects cells and provides a microenvironment that facilitates iHep migration into the liver from the spleen. Bubbles are a lethal factor for surgery and should be avoided completely in the syringe.
3) Correct number of engrafted cells. Overload of cells can also lead to high lethality rate. We recommend engrafting 1–1.5 million iHeps per 25–30 g mouse.
Our protocol also has some limitations: the liver injury induced by irradiation is moderate and single-dose, and the maturation state of our iHeps is not comparable to pHH. Related to both considerations, the degree of chimerism of our model is similar to recent reports describing NOD/Lt-SCID/IL-2Rγ−/− mice or Gunn rats engrafted with iPSC-derived iHeps17,19, but significantly lower than FRG mice or uPA transgenic mice engrafted with pHH. To overcome this caveat, on one hand, one could further optimize the hepatic differentiation protocol to improve the maturation of iHeps. On the other hand, LRG mice could be crossed with FRG mice to generate Ldlr-/-/Fah-/-/Rag2-/-/Il2rg-/- mice.
In summary, here we have described a detailed protocol for generating human liver chimeric animals with FH iHeps, and for testing the functionality of the engrafted iHeps. Importantly, these chimeric mice can be used for in vivo drug testing. Our model can likely be optimized by improving the functionality of iHeps or knocking out additional genes (e.g., Fah) in the recipient LRG mice, and it will be useful to investigate pathological mechanisms of the disease and perform preclinical studies.
The authors have nothing to disclose.
This work was supported by the Shenzhen Science and Technology Council Basic Research Program (JCYJ20150331142757383), Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030502), Hong Kong Research Grant Council Theme Based Research Scheme (T12-705/11), Cooperation Program of the Research Grants Council of the Hong Kong Special Administrative Region and the National Natural Science Foundation of China (N-HKU730/12 and 81261160506), Research Team Project of Guangdong Natural Science Foundation (2014A030312001), Guangzhou Science and Technology Program (201607010086), and Guangdong Province Science and Technology Program (2016B030229007 and 2017B050506007).
Materials | |||
40 µm Cell strainer | BD | B4-VW-352340 | |
6-Well plate | Thermofisher | 140675 | Extracellular matrix coated |
Accutase | Millipore | SCR005 | |
Acetylcholine | Sigma Aldrich | A6625 | Dissolve in water |
Antigen retrieval solution | IHC World | IW-1100-1L | |
Calcium chloride | Sigma Aldrich | C8106 | CaCl2 |
Cell dissociation enzyme | Thermofisher | 12604-013 | TrypLE |
D-glucose | Sigma Aldrich | D8270 | |
Dimethyl sulfoxide | Sigma Aldrich | D5879 | DMSO |
DMEM | Thermofisher | 10829 | Knockout DMEM |
DNase I | Roche | 11284932001 | |
EDTA | USB | 15694 | 0.5 M, PH=8.0 |
Extracellular matrix (for cell suspension) | Corning | 354234 | Matrigel |
Extracellular matrix (for iHep differentiation) | Corning | 354230 | Matrigel |
Hepatocyte basal medium | Lonza | CC-3199 | |
Hepatocyte culture medium | Lonza | CC-3198 | |
High-fat and high-cholesterol diet | Research Diet | D12079B | |
Human Activin A | Peprotech | 120-14E | |
Human hepatocyte growth factor | Peprotech | 100-39 | |
Human iPSC maintenance medium | STEMCELL Technologies | 5850 | mTeSR1 |
Human oncostatin M | Peprotech | 300-10 | |
Ketamine 10% | Alfasan | N/A | |
L-glutamine | Thermofisher | 35050 | |
LDL-C detection kit | WAKO | 993-00404 and 993-00504 | |
Magnesium chloride | VWR | P25108 | MgCl2 |
Meloxicam | Boehringer Ingelheim | NADA 141-213 | |
Monopotassium phosphate | USB | S20227 | KH2PO4 |
Non-essential amino acids | Thermofisher | 11140 | |
PBS | GE | SH30256.02 | Calcium and magnesium-free |
PCSK9 antibodies | Sanofi and Regeneron Pharmaceuticals | SAR236553/REGN727 | Alirocumab |
Phenobarbital | Alfamedic company | 013003 | |
Phenylephrine | RBI | P-133 | Dissolve in water |
Potassium chloride | Sigma Aldrich | P9333 | KCl |
Povidone-iodine | Mundipharma | Betadine | |
Recombinant mouse Wnt3a | R&D Systems | 1324-WN-500/CF | |
ROCK inhibitor Y27632 | Sigma Aldrich | Y0503-5MG | |
RPMI 1640 | Thermofisher | 21875 | |
Serum replacement | Thermofisher | 10828 | |
Silicone coated petri dish | Dow Corning | Sylgard 184 silicone elastomer kit | |
Simvastatin | Merck Sharp & Dohme | ZOCOR | |
Sodium bicarbonate | Sigma Aldrich | S6297 | NaHCO3 |
Sodium chloride | Sigma Aldrich | S7653 | NaCl |
Trypan blue solution 0.4% | Thermofisher | 15250061 | |
U-46619 | Cayman | 16450 | Dissolve in DMSO |
Xylazine 2% | Alfasan | N/A | |
β-mercaptoethanol | Thermofisher | 31350 | |
Name | Company | Catalog Number | Comments |
Antibodies | |||
AAT | DAKO | A0012 | 1:400 |
ALB | Bethyl Laboratories | A80-129 | 1:200 |
ASGPR | Santa Cruz | Sc-28977 | 1:100 |
HNF4A | Santa Cruz | Sc-6557 | 1:35 |
NANOG | Stemgent | 09-0020 | 1:200 |
OCT4 | Stemgent | 09-0023 | 1:200 |
Name | Company | Catalog Number | Comments |
Mice | |||
Il2rg-/- | Jacson lab | 003174 | |
Ldlr-/- | Jacson lab | 002077 | |
Rag2-/- | Jacson lab | 008449 | |
Name | Company | Catalog Number | Comments |
Equipments | |||
Automated cell counter | Invitrogen | Countess | |
Gamma irradiator | MDS Nordion | Gammacell 3000 Elan II | |
Insulin syringe | BD | 324911 | |
Powerlab | ADInstruments | Model 8/30 | |
Slides scanning system | Leica biosystems | Aperio scanScope system | |
Sliding Microtome | Leica biosystems | RM2125RT | |
Stereomicrocope | Nikon | SMZ800 | |
Tissue processing system | Leica biosystems | ASP200S | |
Wire myograph | DMT | 610M | |
Name | Company | Catalog Number | Comments |
Softwares | |||
Digital slide viewing software | Leica | Aperio ImageScope Version 12.3.2 | |
Image J | NIH | Version 1.51e | |
Image processing software | Adobe | Photoshop CC Version 2015 | |
Microscope imaging software | Carl Zeiss | AxioVision LE Version 4.7 |