Name: a familial hypercholesterolemia human liver chimeric mouse model using induced pluripotent stem cell-derived hepatocytes
Uploaded: 2018-09-15T13:00:00.000+00:00
Duration: 10 min 56 s
Description: Watch this Scientific Journal Video about A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes at JoVE.com

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).

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H.-F.T. is the National Coordinator and Investigator of the ODYSSEY OUTCOMES study sponsored by Sanofi and Regeneron Pharmaceuticals.

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&#8211;1.5 million iHeps per 25&#8211;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-2R&#947;&#8722;/&#8722; 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.

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 &#34;humanized&#34; 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.

<table><tbody><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>40 &amp;micro;m Cell strainer</td><td>BD</td><td>B4-VW-352340</td><td></td></tr><tr><td>6-Well plate</td><td>Thermofisher</td><td>140675</td><td>Extracellular matrix coated</td></tr><tr><td>Accutase</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>Acetylcholine</td><td>Sigma Aldrich</td><td>A6625</td><td>Dissolve in water</td></tr><tr><td>Antigen retrieval solution</td><td>IHC World</td><td>IW-1100-1L</td><td></td></tr><tr><td>Calcium chloride</td><td>Sigma Aldrich</td><td>C8106</td><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Cell dissociation enzyme</td><td>Thermofisher</td><td>12604-013</td><td>TrypLE</td></tr><tr><td>D-glucose</td><td>Sigma Aldrich</td><td>D8270</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Sigma Aldrich</td><td>D5879</td><td>DMSO</td></tr><tr><td>DMEM</td><td>Thermofisher</td><td>10829</td><td>Knockout DMEM</td></tr><tr><td>DNase I</td><td>Roche</td><td>11284932001</td><td></td></tr><tr><td>EDTA</td><td>USB</td><td>15694</td><td>0.5 M, PH=8.0</td></tr><tr><td>Extracellular matrix (for cell suspension)</td><td>Corning</td><td>354234</td><td>Matrigel</td></tr><tr><td>Extracellular matrix (for iHep differentiation)</td><td>Corning</td><td>354230</td><td>Matrigel</td></tr><tr><td>Hepatocyte basal medium</td><td>Lonza</td><td>CC-3199</td><td></td></tr><tr><td>Hepatocyte culture medium</td><td>Lonza</td><td>CC-3198</td><td></td></tr><tr><td>High-fat and high-cholesterol diet</td><td>Research Diet</td><td>D12079B</td><td></td></tr><tr><td>Human Activin A</td><td>Peprotech</td><td>120-14E</td><td></td></tr><tr><td>Human hepatocyte growth factor</td><td>Peprotech</td><td>100-39</td><td></td></tr><tr><td>Human iPSC maintenance medium</td><td>STEMCELL Technologies</td><td>5850</td><td>mTeSR1</td></tr><tr><td>Human oncostatin M</td><td>Peprotech</td><td>300-10</td><td></td></tr><tr><td>Ketamine 10%</td><td>Alfasan</td><td>N/A</td><td></td></tr><tr><td>L-glutamine</td><td>Thermofisher</td><td>35050</td><td></td></tr><tr><td>LDL-C detection kit</td><td>WAKO</td><td>993-00404 and 993-00504</td><td></td></tr><tr><td>Magnesium&amp;nbsp;chloride</td><td>VWR</td><td>P25108</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Meloxicam</td><td>Boehringer Ingelheim</td><td>NADA 141-213</td><td></td></tr><tr><td>Monopotassium phosphate</td><td>USB</td><td>S20227</td><td>KH&lt;sub&gt;2&lt;/sub&gt;PO4</td></tr><tr><td>Non-essential amino acids</td><td>Thermofisher</td><td>11140</td><td></td></tr><tr><td>PBS</td><td>GE</td><td>SH30256.02</td><td>Calcium and magnesium-free</td></tr><tr><td>PCSK9 antibodies</td><td>Sanofi and Regeneron Pharmaceuticals</td><td>SAR236553/REGN727</td><td>Alirocumab</td></tr><tr><td>Phenobarbital</td><td>Alfamedic company</td><td>013003</td><td></td></tr><tr><td>Phenylephrine</td><td>RBI</td><td>P-133</td><td>Dissolve in water</td></tr><tr><td>Potassium chloride</td><td>Sigma Aldrich</td><td>P9333</td><td>KCl</td></tr><tr><td>Povidone-iodine</td><td>Mundipharma</td><td>Betadine</td><td></td></tr><tr><td>Recombinant mouse Wnt3a</td><td>R&amp;amp;D Systems</td><td>1324-WN-500/CF</td><td></td></tr><tr><td>ROCK inhibitor Y27632</td><td>Sigma Aldrich</td><td>Y0503-5MG</td><td></td></tr><tr><td>RPMI 1640</td><td>Thermofisher</td><td>21875</td><td></td></tr><tr><td>Serum replacement</td><td>Thermofisher</td><td>10828</td><td></td></tr><tr><td>Silicone coated petri dish</td><td>Dow Corning</td><td>Sylgard 184 silicone elastomer kit</td><td></td></tr><tr><td>Simvastatin</td><td>Merck Sharp &amp;amp; Dohme</td><td>ZOCOR</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>S6297</td><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S7653</td><td>NaCl</td></tr><tr><td>Trypan blue solution 0.4%</td><td>Thermofisher</td><td>15250061</td><td></td></tr><tr><td>U-46619</td><td>Cayman</td><td>16450</td><td>Dissolve in DMSO</td></tr><tr><td>Xylazine 2%</td><td>Alfasan</td><td>N/A</td><td></td></tr><tr><td>&lt;strong&gt;&amp;beta;&lt;/strong&gt;-mercaptoethanol</td><td>Thermofisher</td><td>31350</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AAT</td><td>DAKO</td><td>A0012</td><td>1:400</td></tr><tr><td>ALB</td><td>Bethyl Laboratories</td><td>A80-129</td><td>1:200</td></tr><tr><td>ASGPR</td><td>Santa Cruz</td><td>Sc-28977</td><td>1:100</td></tr><tr><td>HNF4A</td><td>Santa Cruz</td><td>Sc-6557</td><td>1:35</td></tr><tr><td>NANOG</td><td>Stemgent</td><td>09-0020</td><td>1:200</td></tr><tr><td>OCT4</td><td>Stemgent</td><td>09-0023</td><td>1:200</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Mice&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Il2rg&lt;sup&gt;-/- &lt;/sup&gt;&lt;/em&gt;</td><td>Jacson lab</td><td>003174</td><td></td></tr><tr><td>&lt;em&gt;Ldlr&lt;sup&gt;-/- &lt;/sup&gt;&lt;/em&gt;</td><td>Jacson lab</td><td>002077</td><td></td></tr><tr><td>&lt;em&gt;Rag2&lt;sup&gt;-/- &lt;/sup&gt;&lt;/em&gt;</td><td>Jacson lab</td><td>008449</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipments&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Automated cell counter</td><td>Invitrogen</td><td>Countess</td><td></td></tr><tr><td>Gamma irradiator</td><td>MDS Nordion</td><td>Gammacell 3000 Elan II</td><td></td></tr><tr><td>Insulin syringe</td><td>BD</td><td>324911</td><td></td></tr><tr><td>Powerlab</td><td>ADInstruments</td><td>Model 8/30</td><td></td></tr><tr><td>Slides scanning system</td><td>Leica biosystems</td><td>Aperio scanScope system</td><td></td></tr><tr><td>Sliding Microtome</td><td>Leica biosystems</td><td>RM2125RT</td><td></td></tr><tr><td>Stereomicrocope</td><td>Nikon</td><td>SMZ800</td><td></td></tr><tr><td>Tissue processing system</td><td>Leica biosystems</td><td>ASP200S</td><td></td></tr><tr><td>Wire myograph</td><td>DMT</td><td>610M</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Softwares&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Digital slide viewing software</td><td>Leica</td><td>Aperio ImageScope Version 12.3.2</td><td></td></tr><tr><td>Image J</td><td>NIH</td><td>Version 1.51e</td><td></td></tr><tr><td>Image processing software</td><td>Adobe</td><td>Photoshop CC Version 2015</td><td></td></tr><tr><td>Microscope imaging software</td><td>Carl Zeiss</td><td>AxioVision LE Version 4.7</td><td></td></tr></tbody></table>

a familial hypercholesterolemia human liver chimeric mouse model using induced pluripotent stem cell-derived hepatocytes

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.

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&#8211;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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57556/57556fig1.jpg" /> 
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. <a href="/files/ftp_upload/57556/57556fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57556/57556fig2.jpg" /> 
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). <a href="/files/ftp_upload/57556/57556fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57556/57556fig3.jpg" /> 
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. <a href="/files/ftp_upload/57556/57556fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57556/57556fig4.jpg" /> 
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. <a href="/files/ftp_upload/57556/57556fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57556/57556fig5.jpg" /> 
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&#39;s multiple comparison; error bars indicate SEM. Figure 5 is modified from Figure 4A-4C and Figure 5A of our previous report15. <a href="/files/ftp_upload/57556/57556fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes at JoVE.com

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

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 ...

a-familial-hypercholesterolemia-human-liver-chimeric-mouse-model

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.1K Views.  University of Hong Kong-Shenzhen Hospital. Deze methode heeft beantwoord belangrijke vragen in het veld, zoals hoe je een menselijke lever chimeric muis model te genereren, familiale hypercholesterolemie. Het belangrijkste voordeel van deze techniek is dat het mogelijk maakt het uitvoeren van drugstests in vivo. Deze techniek heeft syndicatie voor de behandelingen van familiale hypercholesterolemie.Als een nieuwe therapie voor deze ziekte, het kan worden getest met behulp van dergelijke chimeric dier modellen. Hoewel deze metho...

Video: A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

Cancer Research

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Using Human Induced Pluripotent Stem Cell-derived Hepatocyte-like Cells for Drug Discovery

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn errors in hepatic metabolism. However, to provide a platform that supports the identification of small molecules that can potentially be used to treat liver disease, the procedure requires a culture format that is compatible with screening thousands of compounds. Here, we describe a protocol using completely defined culture conditions, which allow the reproducible differentiation of human iPSCs to hepatocyte-like cells in 96-well tissue culture plates. We also provide an example of using the platform to screen compounds for their ability to lower Apolipoprotein B (APOB) produced from iPSC-derived hepatocytes generated from a familial hypercholesterolemia patient. The availability of a platform that is compatible with drug discovery should allow researchers to identify novel therapeutics for diseases that affect the liver.

The protocol presented here describes a platform for identifying small molecules for the treatment of liver disease. A step-by-step description is presented detailing how to differentiate iPSCs into cells with hepatocyte characteristics in 96-well plates, and to use the cells to screen for small molecules with potential therapeutic activity.

The ability to differentiate human induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) provides new opportunities to study inborn ...

Semi-automated Production of Hepatocyte Like Cells from Pluripotent Stem Cells

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). Current differentiation procedures generate human hepatocyte-like cells (HLCs) displaying a mixture of fetal and adult traits. To improve cell phenotype, we have fully defined our differentiation procedure and the cell niche, resulting in the generation of cell populations which display improved gene expression and&#160;function. While these studies mark progress, the ability to generate large quantities of multi well plates for screening has been limited by labour intensive procedures and batch to batch variation. To tackle this issue, we have developed a semi-automated platform to differentiate pluripotent stem cells into HLCs. Stem cell seeding and differentiation were performed using liquid handling and automatic pipetting systems in&#160;96-well plate format. Following the differentiation, cell phenotype was analyzed using automated microscopy and a multi well luminometer.

This protocol describes a semi-automated approach to produce hepatocyte-like cells from human pluripotent stem cells in a 96 well plate format. This process is rapid and cost-effective, allowing the production of quality assured batches of hepatocyte-like cells for basic and applied human research.

Human pluripotent stem cells represent a renewable source of human tissue. Our research is focused on generating human liver tissue from human embryonic ...

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver failure&#8212;which can be caused by chronic alcohol intake, hepatitis, acute poisoning, or other insults&#8212;is a severe condition that can culminate in bleeding, jaundice, coma, and eventually death. However, approaches to treat liver failure, as well as studies of liver function and disease, have been stymied in part by the lack of a plentiful supply of human liver cells. To this end, this protocol details the efficient differentiation of human pluripotent stem cells (hPSCs) into hepatocyte-like cells, guided by a developmental roadmap that describes how liver fate is specified across six consecutive differentiation steps. By manipulating developmental signaling pathways to promote liver differentiation and to explicitly suppress the formation of unwanted cell fates, this method efficiently generates populations of human liver bud progenitors and hepatocyte-like cells by days 6 and 18 of PSC differentiation, respectively. This is achieved through the temporally-precise control of developmental signaling pathways, exerted by small molecules and growth factors in a serum-free culture medium. Differentiation in this system occurs in monolayers and yields hepatocyte-like cells that express characteristic hepatocyte enzymes and have the ability to engraft a mouse model of chronic liver failure. The ability to efficiently generate large numbers of human liver cells in vitro has ramifications for treatment of liver failure, for drug screening, and for mechanistic studies of liver disease.

This protocol details a monolayer, serum-free method to efficiently generate hepatocyte-like cells from human pluripotent stem cells (hPSCs) in 18 days. This entails six steps as hPSCs sequentially differentiate into intermediate cell-types such as the primitive streak, definitive endoderm, posterior foregut and liver bud progenitors before forming hepatocyte-like cells.

The liver detoxifies harmful substances, secretes vital proteins, and executes key metabolic activities, thus sustaining life. Consequently, liver ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Biology

Generation of a Humanized Mouse Liver Using Human Hepatic Stem Cells

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in which the liver has been repopulated with functional human hepatocytes, could serve as a useful tool for investigating human hepatic cell biology, drug metabolism, and other preclinical applications. One of the key factors required for successful transplantation of human hepatocytes into mice is the elimination of the endogenous hepatocytes to prevent competition with the human cells and provide a suitable space and microenvironment for promoting human donor cell expansion and differentiation. To date, two major liver injury mouse models utilizing fumarylacetoacetate hydrolase (Fah) and uroplasminogen activator (uPA) mice have been established. However, Fah mice are used mainly with mature hepatocytes and the application of the uPA model is limited by decreased breeding. To overcome these limitations, Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice were used for in vivo differentiation of immature human hepatocytes and humanized liver generation. Human hepatic stem cells (HpSCs) successfully repopulated the livers of Alb-TRECK/SCID mice that had developed lethal fulminant hepatic failure following diphtheria toxin (DT) treatment. This model of a humanized liver in Alb-TRECK/SCID mice will have functional applications in studies involving drug metabolism and drug-drug interactions and will promote other in vivo and in vitro studies.

Here, we present a novel humanized mouse liver model generated in Alb-toxin receptor mediated cell knockout (TRECK)/SCID mice following the transplantation of immature and expandable human hepatic stem cells.

A novel animal model involving chimeric mice with humanized livers established via human hepatocyte transplantation has been developed. These mice, in ...

Medicine

Integrative Protocol for Generation of iPSC-Derived 3D Human Hepatic Organoids

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing human induced pluripotent stem cells (hiPSCs) to generate 3D cultures of human hepatic organoids (HHOs). The utilization of HHOs offers a valuable approach to understanding liver development, unraveling liver diseases, conducting high-throughput studies for drug development, and exploring the potential for liver transplantation. In the former investigation, through immunofluorescence and quantitative RT-PCR techniques, the progression was monitored, identifying the presence of various cell populations, such as hepatoblasts and the two types of hepatoblast-derived cells: cholangiocytes or hepatocyte-like cells, across different developmental stages. This report presents a straightforward 3D protocol starting from hiPSC to acquire HHOs that mirror the stages of human embryo development. The protocol, spanning 46-50 days, encompasses several steps: (i) meticulous management of hiPSC culture to generate HHOs, (ii) initiation of cell differentiation in 2D and the subsequent transition to 3D, and (iii) an optimized dissociation strategy to break down HHOs into single cells for single-cell RNA sequencing. As an illustration of the broad applications of this approach, the present protocol was previously applied to unravel the role of thyroid hormone signaling in developing liver cells.

Human iPSC-derived 3D hepatic organoids constitute a potential tool for understanding the thyroid hormone's action on liver development.

Obtaining stable hepatic cells in culture poses a significant challenge for liver studies. Bearing this in mind, an optimized method is depicted utilizing ...

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 &#215; 106 cells per liver and cell viability of &#62;85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of &#62;35% in mouse hepatocytes.
To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.

This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation.

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 ...

Bioengineering

An Efficient Method for Directed Hepatocyte-Like Cell Induction from Human Embryonic Stem Cells

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug screening applications. Provided here is an efficient and reproducible method for differentiation of hESCs into functional HLCs. The establishment of an endoderm lineage is a key step in the differentiation to HLCs. By our method, we regulate the key signaling pathways by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE), followed by generation of hepatic progenitor cells, and finally HLCs with typical hepatocyte morphology in a stagewise method with completely defined reagents. The hESC-derived HLCs produced by this method express stage-specific markers (including albumin, HNF4&#945; nuclear receptor, and sodium taurocholate cotransporting polypeptide (NTCP)), and show special characteristics related to mature and functional hepatocytes (including indocyanine green staining, glycogen storage, hematoxylin-eosin staining and CYP3 activity), and can provide a platform for the development of HLC-based applications in the study of liver diseases.

This manuscript describes a detailed protocol for differentiation of human embryonic stem cells (hESCs) into functional hepatocyte-like cells (HLCs) by continuously supplementing Activin A and CHIR99021 during hESC differentiation into definitive endoderm (DE).

The potential functions of hepatocyte-like cells (HLCs) derived from human embryonic stem cells (hESCs) hold great promise for disease modeling and drug ...

A Familial Hypercholesterolemia Human Liver Chimeric Mouse Model Using Induced Pluripotent Stem Cell-derived Hepatocytes

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