Name: a familial hypercholesterolemia human liver chimeric mouse model using induced pluripotent stem cell-derived hepatocytes
Uploaded: 2018-09-15T13:00:00
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).

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
<ol>
	<li>Generation of immunodeficient Ldlr knockout (KO) mice.
	<ol>
		<li>Use the mice strains Ldlr-/-, Rag2-/-, and Il2rg-/- (see Table of Materials).</li>
	...

<ol>
	<li>Brown, M. S., Goldstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+receptor-mediated+pathway+for+cholesterol+homeostasis.">A receptor-mediated pathway for cholesterol homeostasis.</a> Science. 232 (4746), 34-47 (1986).</li><li>Endo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+discovery+and+development+of+HMG-CoA+reductase+inhibitors.">The discovery and development of HMG-CoA reductase inhibitors.</a> J Lipid Res. 33 (11), 1569-1582 (1992).</li><li>Dubuc, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Statins+upregulate+PCSK9,+the+gene+encoding+the+proprotein+convertase+neural+apoptosis-regulated+convertase-1+implicated+in+familial+hypercholesterolemia.">Statins upregulate PCSK9, the gene encoding the proprotein convertase neural apoptosis-regulated convertase-1 implicated in familial hypercholesterolemia.</a> Arterioscler Thrombo Vasc Biol. 24 (8), 1454-1459 (2004).</li><li>Robinson, J. 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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+liver+chimeric+mice+provide+a+model+for+hepatitis+B+and+C+virus+infection+and+treatment.">Human liver chimeric mice provide a model for hepatitis B and C virus infection and treatment.</a> J Clin Invest. 120 (3), 924-930 (2010).</li><li>Azuma, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+expansion+of+human+hepatocytes+in+Fah(-/-)/Rag2(-/-)/Il2rg(-/-)+mice.">Robust expansion of human hepatocytes in Fah(-/-)/Rag2(-/-)/Il2rg(-/-) mice.</a> Nat Biotechnol. 25 (8), 903-910 (2007).</li><li>Bissig-Choisat, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+rescue+of+human+familial+hypercholesterolaemia+in+a+xenograft+mouse+model.">Development and rescue of human familial hypercholesterolaemia in a xenograft mouse model.</a> Nat Commun. 6, 7339 (2015).</li><li>Yang, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+human+liver+chimeric+mice+with+hepatocytes+from+familial+hypercholesterolemia+induced+pluripotent+stem+cells.">Generation of human liver chimeric mice with hepatocytes from familial hypercholesterolemia induced pluripotent stem cells.</a> Stem Cell Rep. 8 (3), 605-618 (2017).</li><li>Kajiwara, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Donor-dependent+variations+in+hepatic+differentiation+from+human-induced+pluripotent+stem+cells.">Donor-dependent variations in hepatic differentiation from human-induced pluripotent stem cells.</a> Proc Natl Acad Sci USA. 109 (31), 12538-12543 (2012).</li><li>Chen, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amelioration+of+hyperbilirubinemia+in+gunn+rats+after+transplantation+of+human+induced+pluripotent+stem+cell-derived+hepatocytes.">Amelioration of hyperbilirubinemia in gunn rats after transplantation of human induced pluripotent stem cell-derived hepatocytes.</a> Stem Cell Rep. 5 (1), 22-30 (2015).</li><li>Ortmann, D., Vallier, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variability+of+human+pluripotent+stem+cell+lines.">Variability of human pluripotent stem cell lines.</a> Curr Opin Genet Dev. 46, 179-185 (2017).</li><li>Liu, H., Kim, Y., Sharkis, S., Marchionni, L., Jang, Y. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+liver+regeneration+potential+of+human+induced+pluripotent+stem+cells+from+diverse+origins.">In vivo liver regeneration potential of human induced pluripotent stem cells from diverse origins.</a> Sci Transl Med. 3 (82), 82ra39 (2011).</li></ol>

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...

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>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;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>CaCl2</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&#160;chloride</td>
			<td>VWR</td>
			<td>P25108</td>
			<td>MgCl2</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>KH2PO4</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&#38;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 &#38; Dohme</td>
			<td>ZOCOR</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S6297</td>
			<td>NaHCO3</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>&#946;-mercaptoethanol</td>
			<td>Thermofisher</td>
			<td>31350</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Il2rg-/- </td>
			<td>Jacson lab</td>
			<td>003174</td>
			<td></td>
		</tr>
		<tr>
			<td>Ldlr-/- </td>
			<td>Jacson lab</td>
			<td>002077</td>
			<td></td>
		</tr>
		<tr>
			<td>Rag2-/- </td>
			<td>Jacson lab</td>
			<td>008449</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipments</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Softwares</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...

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 ...

This method have answered key questions in the field, such as how to generate a human liver chimeric mouse model, familial hypercholesterolemia. The main advantage of this technique is that it allows performing drug tests in in vivo. This technique has syndication for the treatments of familial hypercholesterolemia.As a new therapy for this disease, it can be tested using such chimeric animal models. Though this method can provide insight into familial hypercholesterolemia, it can also be applied to other inherited liver diseases. 24 hours prior to engraftment, thaw 40 microliters of extracellular matrix per mouse, by placing in an icebox, in a cold room.Chill an insulin syringe for each mouse to be injected and a box of 200 microliter tips in a four degree Celsius refrigerator. One hour prior to engraftment, warm the cell dissociation enzyme, supplemented with 50 micrograms per milliliter of DNAase 1, to room temperature and place RPMI 1640 medium, supplemented with 20 percent serum replacement on ice. Take phase contrast images of iHeps to record their status, including cell morphology, growth and cell density.Next, wash each well of iHeps with 2 milliliters of room temperature calcium and magnesium ion-free PBS twice and then add one milliliter of the pre-warmed cell dissociation enzyme to each well. Return the cells to the incubator for eight to ten minutes. Monitor the cell morphology under the microscope.When most of the cells become round, add in equal volume of cold medium per well, pipette the cells gently to detach from the plate, and transfer the cell suspension to a new 15 milliliter tube. This step is critical for the hepatocytes reliability. If the cells are difficult to detach from the plate, some can be left on the plate.And if the cells detach, Then pipette gently after centrifugation, to obtain a single-cell suspension. Repeat the cell-detachment procedure for each well until nearly all attached cells are collected. Then centrifuge at 200 g for three minutes at four degrees celsius.Following centrifugation, remove the supernatant and re-suspend the cells with two milliliters of cold PBS in the fifteen milliliter tube. Pipette the cells gently to obtain a single-cell suspension. Then, pass the cells through a 40 micron cell strainer to remove aggregates.Normally, one to two million cells about can be harvested after few-ter-ing. Next, add microliters of 0.4 percent Trypan blue solution to 20 microliters of cell suspension. Count the cells and record the concentration of cell suspension as C.Calculate the required volume of cell suspension to inject one million cells per mouse.Then allocate the required volume of cell suspension into 15 milliliter tubes and centrifuge at 200 g for three minutes at four degrees Celsius. After removing the supernatant, re-suspend the cells in 27.5 microliters of cold PBS per mouse. And then add an equal volume of extracellular matrix for a final volume of 55 microliters per mouse.Now, place one of the cold insulin syringes on ice. Unplug the piston, transfer 55 microliters of cell suspension into the syringe and then put the piston back. Discharge bubbles carefully, and put the syringe back on ice.Ensure that LRG mice are properly anesthetized before beginning the injection procedure. Apply veterinary ointment over the eyes to prevent dryness during the anesthesia procedure. Once the mouse has lost muscle reflex to stimulation, place it in the right, lateral, decubitus position.Apply a thick layer of depilatory cream to the incision area in the left flank, and leave for five to eight minutes. Remove the depilatory cream and hair by wiping the area with a water-moistened gauze pad. Scrub the left flank with 70 percent ethanol three times.Then locate the spleen, which can be seen in the left flank. Followed by a final soaking with betadine for disinfection. After using scissors to make a 0.5 to one centimeter incision in the skin and abdominal wall, use forceps to exteriorize the spleen by gently pulling the surrounding adipose tissue.Gently stabilize the spleen using a swab. Insert the needle of the insulin syringe three to four millimeters into the parenchyma of the spleen, and gently inject approximately 50 microliters of cell suspension. Retract the needle and place a cotton swab over the injection for one minute to prevent bleeding and spillage of material.Return the spleen to the peritoneum, and close the wound with five-oh nylon sutures. After suturing, place the mouse in a clean cage with easy access to food and water. Begin by removing the internal organs from the freshly euthanized mouse so that the descending aorta, parallel to the spine, is visible.Then dissect away the adjacent tissue, and remove the heart. Use fine scissors to dissect the aortae, placing each one into cold, oxygenated Krebs solution. After collection, transfer the aortae in Krebs solution to a silicone-coated Petri dish.Pin the connective tissue to fix the aorta position without stretching it. Under a sterile microscope, use fine forceps and spring scissors to dissect the aorta free from the surrounding fat and adventitial tissue without damaging the vessel wall. Then cut each aorta into one and a half to two millimeter length segments.Next, cut a two centimeter long piece of 40 micron thick stainless wire, and gently insert into the aorta lumen. Hold the wire to transfer the segment to the wire myograph chamber, filled with oxygenated Krebs solution. To measure the length of the aortae segments when studying contractility, place each segment perpendicularly in between the jaws, and record the D1 reading on the micrometer.Then remove the segment and move the jaws together. Record the D0 reading and calculate the length of the segment. Next, clamp the wire and secure it with the screwdriver whilst placing the unstretched segment in between the jaws.For normalization before the experiment, set the myograph to zero in the unstretched position. Then slowly move the jaws apart and observe the aorta tension change until reaching three millinewton. After 15 minutes, drain the solution from the myograph chamber and replace with fresh Krebs solution.After waiting for another 15 minutes, again adjust the tension to three millinewton. Then change the standard Krebs solution to Krebs solution supplemented with 60 millimolar potassium chloride, to induce contraction for at least fifteen minutes. Rinse with fresh Krebs solution three times, then add increasing concentrations of phenylephrine.For example, 10 nanomolar to 100 micromolar. Then, after washing out with standard Krebs solution, add a single concentration of phenylephrine, at about 70 percent of maximal contraction. When the contraction is stable, add increasing concentrations of acetylcholine at two-minute intervals.For example, one to three nanomolar to ten to thirty micromolar to induce vasodilation. This image shows a representative whole-section scanned image of human albumin staining in a mouse liver repopulated with low-density lipoprotein receptor-deficient iHeps. Arrows indicate clusters of human iHeps engrafted into the mouse liver.The clusters of engrafted human iHeps are seen in more detail here. This scatterplot graph shows the percentage of repopulated human albumin positive cells corresponding to iHep containing areas in the mouse liver, from different donor iPSCs. This is a representative image of immunohistochemical staining for human nuclei in a mouse liver, with engrafted familial hypercholesterolemia iHeps.The bar graph shows the percentage of repopulated human nuclei positive iHeps from different donor iPSCs in LRG mouse livers. These are representative images of human albumin and human nuclei staining on two consecutive sections of a mouse liver, repopulated with wild-type iHeps. Finally, this image shows endothelium-dependent vasodilation of the aorta in familial hypercholesterolemia human liver chimeric mice, in response to increasing concentrations of acetylcholine.After watching this video, you should have a good understanding of how to generate human liver chimeric mice, using human iPSC-derived hepatocytes.

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

Research

JoVE Journal

Developmental Biology

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis 1. The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced&#160;pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced&#160;pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Dendritic spines of pyramidal neurons are the sites of most excitatory synapses in mammalian brain cortex. This method describes a 3D quantitative analysis of spine morphologies in human cortical pyramidal glutamatergic neurons derived from induced&#160;pluripotent stem cells.

Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

Differentiating Chondrocytes from Peripheral Blood-derived Human Induced Pluripotent Stem Cells

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an integration-free method. Following embryoid body (EB) formation and fibroblastic cell expansion, the iPSCs are induced for chondrogenic differentiation for 21 days under serum-free and xeno-free conditions. After chondrocyte induction, the phenotypes of the cells are evaluated by morphological, immunohistochemical, and biochemical analyses, as well as by the quantitative real-time PCR examination of chondrogenic differentiation markers. The chondrogenic pellets show positive alcian blue and toluidine blue staining. The immunohistochemistry of collagen II and X staining is also positive. The sulfated glycosaminoglycan (sGAG) content and the chondrogenic differentiation markers COLLAGEN 2 (COL2), COLLAGEN 10 (COL10), SOX9, and AGGRECAN are significantly upregulated in chondrogenic pellets compared to hiPSCs and fibroblastic cells. These results suggest that PBCs can be used as seed cells to generate iPSCs for cartilage repair, which is patient-specific and cost-effective.

We present a protocol to generate a chondrogenic lineage from human peripheral blood (PB) via induced pluripotent stem cells (iPSCs) using an integration-free method, which includes embryoid body (EB) formation, fibroblastic cells expansion, and chondrogenic induction.

In this study, we used peripheral blood cells (PBCs) as seed cells to produce chondrocytes via induced pluripotent stem cells (iPSCs) in an ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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 ...

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells (hiPSC-ECs)

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected tissues via a well-characterized cascade of pro-adhesive receptors which are upregulated on the leukocyte cell surface upon the inflammatory trigger. Inflammatory responses differ between individuals in the population and the genetic background can contribute to these differences. Human induced pluripotent stem cells (hiPSCs) have been shown to be a reliable source of ECs (hiPSC-ECs), thus representing an unlimited source of cells that capture the genetic identity and any genetic variants or mutations of the donor. hiPSC-ECs can therefore be used for modeling inflammatory responses in donor-specific cells. Inflammatory responses can be modeled by determining leukocyte adhesion to the hiPSC-ECs under physiological flow. This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Microfluidic Assay for the Assessment of Leukocyte Adhesion to Human Induced Pluripotent Stem Cell-derived Endothelial Cells hiPSC-ECs

This step-by-step protocol provides a detailed description of the experimental setup and data analysis for the assessment of inflammatory responses in hiPSC-ECs and the analysis of leukocyte adhesion under physiological flow.

Endothelial cells (ECs) are essential for the regulation of inflammatory responses by either limiting or facilitating leukocyte recruitment into affected ...