Name: efficient differentiation of human pluripotent stem cells into liver cells
Uploaded: 2019-06-11T13:30:00
Description: Watch this Scientific Journal Video about Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells at JoVE.com

We thank Bing Lim&#160;for discussions and the Stanford Institute for Stem Cell Biology &#38; Regenerative Medicine for infrastructure support. This work was supported by the California Institute for Regenerative Medicine (DISC2-10679) and the Stanford-UC Berkeley Siebel Stem Cell Institute (to L.T.A. and K.M.L.) and the Stanford Beckman Center for Molecular and Genetic Medicine as well as the Anonymous, Baxter and DiGenova families (to K.M.L.).

1. Preparation of Differentiation Media
NOTE: Refer to the Table of Materials for manufacturer information regarding the materials and reagents used.
<ol>
	<li>Preparation of base chemically defined media (CDM) 
	NOTE: CDM2, CDM3, CDM4 and CDM5 are chemically defined media that are used as base medias for differentiating hPSCs to liver cells at various stages. The composition of these medias can be found in Table 1.
	<o...

<ol>
	<li>Bernal, W., Wendon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+Liver+Failure.">Acute Liver Failure.</a> New England Journal of Medicine. 369, 2525-2534 (2013).</li><li>Ang, L. T., 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=A+Roadmap+for+Human+Liver+Differentiation+from+Pluripotent+Stem+Cells.">A Roadmap for Human Liver Differentiation from Pluripotent Stem Cells.</a> Cell Reports. 22, 2190-2205 (2018).</li><li>Fisher, R. A., Strom, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Hepatocyte+Transplantation:+Worldwide+Results.">Human Hepatocyte Transplantation: Worldwide Results.</a> Transplantation. 82, 441-449 (2006).</li><li>Dhawan, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+human+hepatocyte+transplantation:+Current+status+and+challenges.">Clinical human hepatocyte transplantation: Current status and challenges.</a> Liver transplantation. 21, S39-S44 (2015).</li><li>Salama, 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=Autologous+Hematopoietic+Stem+Cell+Transplantation+in+48+Patients+with+End-Stage+Chronic+Liver+Diseases.">Autologous Hematopoietic Stem Cell Transplantation in 48 Patients with End-Stage Chronic Liver Diseases.</a> Cell Transplantation. 19, 1475-1486 (2017).</li><li>Tan, A. K. Y., Loh, K. M., Ang, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+regenerative+potential+and+functionality+of+human+liver+cells+in+mice.">Evaluating the regenerative potential and functionality of human liver cells in mice.</a> Differentiation. 98, 25-34 (2017).</li><li>Loh, K. 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=Efficient+Endoderm+Induction+from+Human+Pluripotent+Stem+Cells+by+Logically+Directing+Signals+Controlling+Lineage+Bifurcations.">Efficient Endoderm Induction from Human Pluripotent Stem Cells by Logically Directing Signals Controlling Lineage Bifurcations.</a> Cell Stem Cell. 14, 237-252 (2014).</li><li>Loh, K. 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=Mapping+the+Pairwise+Choices+Leading+from+Pluripotency+to+Human+Bone,+Heart,+and+Other+Mesoderm+Cell+Types.">Mapping the Pairwise Choices Leading from Pluripotency to Human Bone, Heart, and Other Mesoderm Cell Types.</a> Cell. , 451-467 (2016).</li><li>Zhao, 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=Promotion+of+the+efficient+metabolic+maturation+of+human+pluripotent+stem+cell-derived+hepatocytes+by+correcting+specification+defects.">Promotion of the efficient metabolic maturation of human pluripotent stem cell-derived hepatocytes by correcting specification defects.</a> Cell Research. 23, 157-161 (2012).</li><li>Si Tayeb, K., 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=Highly+efficient+generation+of+human+hepatocyte-like+cells+from+induced+pluripotent+stem+cells.">Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells.</a> Hepatology. 51, 297-305 (2010).</li><li>Carpentier, A., 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=Hepatic+differentiation+of+human+pluripotent+stem+cells+in+miniaturized+format+suitable+for+high-throughput+screen.">Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen.</a> Stem Cell Research. 16, 640-650 (2016).</li><li>Avior, 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=Microbial-derived+lithocholic+acid+and+vitamin+K2+drive+the+metabolic+maturation+of+pluripotent+stem+cells-derived+and+fetal+hepatocytes.">Microbial-derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent stem cells-derived and fetal hepatocytes.</a> Hepatology. 62, 265-278 (2015).</li><li>Ogawa, S., 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=Three-dimensional+culture+and+cAMP+signaling+promote+the+maturation+of+human+pluripotent+stem+cell-derived+hepatocytes.">Three-dimensional culture and cAMP signaling promote the maturation of human pluripotent stem cell-derived hepatocytes.</a> Development. 140, 3285-3296 (2013).</li><li>Rostovskaya, M., Bredenkamp, N., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+consistent+generation+of+pancreatic+lineage+progenitors+from+human+pluripotent+stem+cells.">Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells.</a> Philosophical Transactions of the Royal Society B: Biological Sciences. 370, 20140365 (2015).</li><li>Haridass, 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=Repopulation+Efficiencies+of+Adult+Hepatocytes,+Fetal+Liver+Progenitor+Cells,+and+Embryonic+Stem+Cell-Derived+Hepatic+Cells+in+Albumin-Promoter-Enhancer+Urokinase-Type+Plasminogen+Activator+Mice.">Repopulation Efficiencies of Adult Hepatocytes, Fetal Liver Progenitor Cells, and Embryonic Stem Cell-Derived Hepatic Cells in Albumin-Promoter-Enhancer Urokinase-Type Plasminogen Activator Mice.</a> The American Journal of Pathology. 175, 1483-1492 (2009).</li></ol>

This method enables the generation of enriched populations of liver bud progenitors, and subsequently hepatocyte-like cells, from hPSCs. The ability to generate enriched populations of human liver cells is important for the practical utilization of such cells. Previous methods to generate hepatocytes from hPSCs yielded impure cell populations containing both liver and non-liver cells that, upon transplantation into rodents, yielded bone and cartilage in addition to liver tissue15. Hence the explic...

The purpose of this protocol is to efficiently differentiate human pluripotent stem cells (hPSCs) into enriched populations of liver bud progenitors and hepatocyte-like cells2. Access to a ready supply of human liver progenitors and hepatocyte-like cells will accelerate efforts to investigate liver function and disease and could enable new cellular transplantation therapies for liver failure3,4,5. This has proven challenging in the past since hPSCs (which include embryonic and induced pluripotent stem cells) can differentiate into all the cell-types of the human body; consequently, it has been difficult to exclusively differentiate them into a pure population of a single cell-type, such as liver cells6.
To precisely differentiate hPSCs into liver cells, first it is critical to understand not only how liver cells are specified but also how non-liver cell-types develop. Knowledge of how non-liver cells develop is important to logically suppress the formation of non-liver lineages during differentiation, thereby exclusively guiding hPSCs towards a liver fate2. Second, it is essential to delineate the multiple developmental steps through which hPSCs differentiate towards a liver fate. It is known that hPSCs sequentially differentiate into multiple cell-types known as the primitive streak (APS), definitive endoderm (DE), posterior foregut (PFG) and liver bud progenitors (LB) before forming hepatocyte-like cells (HEP). Earlier work revealed the signals specifying liver fate and the signals that suppressed the formation of alternate non-liver cell-types (including stomach, pancreatic, and intestinal progenitors) at each developmental lineage choice2,7,8.
Collectively, these insights have given rise to a serum-free, monolayer method to differentiate hPSCs towards primitive streak, definitive endoderm, posterior foregut, liver bud progenitors and finally, hepatocyte-like cells2. Overall the method involves the seeding of hPSCs in a monolayer at an appropriate density, preparing six cocktails of differentiation media (containing growth factors and small molecules that regulate various developmental signaling pathways), and sequentially adding these media to induce differentiation over the course of 18 days. During the process, no passaging of cells is needed. Of note, because this method explicitly includes signals that suppress the formation of non-liver cell-types, this differentiation approach1 more efficiently generates liver progenitors and hepatocyte-like cells by comparison to extant differentiation methods2,9,10,11,12. Furthermore, the protocol described in this text enables the faster generation of hepatocytes that ultimately express higher levels of hepatic transcription factors and enzymes than those produced by other protocols9,10,11,12.
The protocol described here has certain advantages over current differentiation protocols. First, it entails monolayer differentiation of hPSCs, which is technically simpler compared to three-dimensional differentiation methods, such as those that rely on embryoid bodies13. Second,&#160;this method exploits a recent advance whereby definitive endoderm cells (an early precursor to liver cells) can be efficiently and rapidly generated within 2 days of hPSC differentiation2,7, thus enabling the subsequent production of hepatocytes with increased purity. Third, in side-by-side comparisons, the hepatocyte-like cells produced by this method2 produce more ALBUMIN&#160;and express higher levels of hepatic transcription factors and enzymes compared to hepatocytes produced in other methods10,11,12.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >&#160;Geltrex</td>
			<td >Thermofisher Scientific</td>
			<td >A1569601</td>
			<td ></td>
		</tr>
		<tr >
			<td >1:1 DMEM/F12</td>
			<td>Gibco</td>
			<td>11320033</td>
			<td></td>
		</tr>
		<tr >
			<td >0.2&#160;&#956;m pore membrane filter</td>
			<td>Millipore</td>
			<td>GTTP02500</td>
			<td ></td>
		</tr>
		<tr >
			<td >mTeSR1</td>
			<td>Stem Cell Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr >
			<td >Thiazovivin</td>
			<td>Tocris Bioscience</td>
			<td>3845</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;Accutase</td>
			<td>Gibco or Millipore&#160;</td>
			<td>Gibco A11105, Millipore&#160; SCR005</td>
			<td></td>
		</tr>
		<tr >
			<td >IMDM, GlutaMAX&#8482; Supplement</td>
			<td>Thermofisher Scientific</td>
			<td>31980030</td>
			<td></td>
		</tr>
		<tr >
			<td >Ham&#39;s F-12 Nutrient Mix, GlutaMAX&#8482; Supplement</td>
			<td>Thermofisher Scientific</td>
			<td>31765035</td>
			<td></td>
		</tr>
		<tr >
			<td >KOSR, Knockout serum replacement</td>
			<td>Thermofisher Scientific</td>
			<td>10828028</td>
			<td></td>
		</tr>
		<tr >
			<td >Poly(vinyl alcohol)</td>
			<td>Sigma-Aldrich&#160;&#160;</td>
			<td>P8136</td>
			<td></td>
		</tr>
		<tr >
			<td >Transferrin&#160;</td>
			<td >Sigma-Aldrich&#160;&#160;</td>
			<td >10652202001</td>
			<td></td>
		</tr>
		<tr >
			<td >Chemically Defined Lipid Concentrate</td>
			<td>Thermofisher Scientific</td>
			<td >11905031</td>
			<td></td>
		</tr>
		<tr >
			<td >human Activin</td>
			<td >R&#38;D</td>
			<td>338-AC</td>
			<td></td>
		</tr>
		<tr >
			<td >CHIR99201</td>
			<td >Tocris</td>
			<td>4423</td>
			<td></td>
		</tr>
		<tr >
			<td >PI103</td>
			<td >Tocris</td>
			<td >2930/1</td>
			<td></td>
		</tr>
		<tr >
			<td >human FGF2</td>
			<td >R&#38;D</td>
			<td>233-FB</td>
			<td></td>
		</tr>
		<tr >
			<td >DM3189</td>
			<td >Tocris</td>
			<td>6053/10</td>
			<td></td>
		</tr>
		<tr >
			<td >A83-01</td>
			<td >Tocris</td>
			<td>2939/10</td>
			<td></td>
		</tr>
		<tr >
			<td >Human BMP4</td>
			<td >R&#38;D</td>
			<td>314-BP</td>
			<td></td>
		</tr>
		<tr >
			<td >C59</td>
			<td >Tocris</td>
			<td>5148</td>
			<td></td>
		</tr>
		<tr >
			<td >TTNPB</td>
			<td >Tocris</td>
			<td>0761/10</td>
			<td></td>
		</tr>
		<tr >
			<td >Forskolin</td>
			<td >Tocris</td>
			<td>1099/10</td>
			<td></td>
		</tr>
		<tr >
			<td >Oncostatin M</td>
			<td >R&#38;D</td>
			<td>295-OM</td>
			<td></td>
		</tr>
		<tr >
			<td >Dexamethasone</td>
			<td >Tocris</td>
			<td>1126</td>
			<td></td>
		</tr>
		<tr >
			<td >Ro4929097</td>
			<td >Selleck Chem</td>
			<td>S1575</td>
			<td></td>
		</tr>
		<tr >
			<td >AA2P</td>
			<td>Cayman chemicals</td>
			<td>16457</td>
			<td></td>
		</tr>
		<tr >
			<td >human recombinant Insulin</td>
			<td>Sigma-Aldrich&#160;&#160;</td>
			<td>11061-68-0</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

After 24 h of APS differentiation, colonies will generally adopt a different morphology than undifferentiated colonies concomitant with a loss of the bright border that typically circumscribes hPSC colonies. Morphologically, primitive streak cells generally have ragged borders and are more spread and less compact than hPSCs-this is evocative of an epithelial-to-mesenchymal transition as pluripotent epiblast cells differentiate and ingress into the primitive streak in vivo. If the colony s...

Watch this Scientific Journal Video about Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells at JoVE.com

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

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 ...

This method enables the generation of large numbers of human liver bud progenitors and hepatocyte-like cells with high purity. The ability to generate these liver cells could be important for regenerative medicine and other fields. By controlling the developmental signaling pathways to promote liver differentiation and suppress the formation of unwanted cell types, this method enables efficient generation of human liver bud progenitors and hepatocyte-like cells by six and 18 days of differentiation respectively.The main advantage of this technique is the high purity of the human liver cells generated. To being this procedure, add 50 milliliters of IMDM to a conical flask containing a stir bar. Heat the medium to 50 degrees Celsius and add 0.5 grams of PVA while continuously stirring to prepare a PVA stock at a concentration of 10 milligrams per milliliter.After this, remove the PVA solution from the heating pad and allow it to cool down to room temperature. Once the solution is cooled, filter it through a sterile, 0.2 micrometer filter. Prepare the CDM2 and CDM3 by combining the filtered PVA with other reagents as outlined in table one of the text protocol.Use a sterile, 0.2 micrometer filter to filter all media. Then, prepare the remaining base media, CDM4 and CDM5, as outlined in table one of the text protocol. To prepare the differentiation medium, first thaw the frozen small molecules and/or growth factors at room temperature.Aliquot out the required amount of base medium and add the specified small molecules and growth factors to the base medium at the appropriate concentrations. The day before use, thaw the matrix at four degrees Celsius. The next day, dilute the matrix at a ratio of one to 100 by adding 500 microliters of matrix into 50 milliliters of cold DMEM.Pipette the diluted matrix into the required number of wells using just enough volume of solution to cover the surface of the well. Transfer the matrix-coated plate to an incubator at 37 degrees Celsius for at least 60 minutes. Immediately prior to seeding, aspirate the remaining matrix solution from the coated wells.Then, aspirate the medium from a largely confluent hPSC culture plate and add commercially-bought dissociation agent, making sure to use just enough to cover the surface on which the cells are growing. Incubate the hPSCs in the dissociation agent at 37 degrees Celsius for five minutes or until some colonies begin detaching. Next, add DMEM and F-12 to dilute the dissociation agent.Use a five milliliter serological pipette to gently pipette up and down multiple times to wash off all cells from the surface of the well. Collect the resuspended single cells in a 50 milliliter conical tube and dilute with DMEM and F-12 as needed. Centrifuge this tube of collected hPSCs at 350 times g and at four degrees Celsius for three minutes to pellet the cells.While waiting for the centrifugation, aspirate the matrix from the plate in which the hPSCs will be seeded. Add a sufficient amount of mTeSR media to recipient wells to cover them. When the centrifugation is complete, carefully aspirate the supernatant, leaving the pelleted hPSCs at the bottom of the conical tube.Resuspend the cell pellet in mTeSR supplemented with one micromolar of commercially-obtained Thiazovivin. Using a P1000 pipette, gently triturate two to three times to evenly resuspend the cell pellet into a single cell suspension. Immediately pipette 10 microliters of the cell suspension into a hemocytometer and count the number of cells.Adjust the volume of the resuspended hPSCs with Thiazovivin-supplemented mTeSR to achieve the desired cell concentration for plating. Then, shake the plate in a cross pattern several times to make sure that the cells are evenly distributed. After the hPSCs have been plated for at least 24 hours, use a phase contrast microscope to check the morphology of the cells with specific emphasis on the diameter of plated hPSC colonies.Next, prepare day one APS differentiation medium as outlined in table two of the text protocol. One day after seeding, aspirate the Thiazovivin-supplemented mTeSR from the plated hPSCs and briefly wash them with IMDM media. After this, add day one medium to the hPSCs.Record the time and place the cells back into the incubator at 37 degrees Celsius. Continue with the subsequent differentiation steps by preparing the needed differentiation medium and adding it to the cells on the respective days of differentiation around the same time of day. In this study, enriched populations of liver bud progenitors and subsequently hepatocyte-like cells are generated from hPSCs.By day two of differentiation, primitive streak cells have differentiated into day two definitive endoderm cells. The vast majority of these cells express SOX17 and FOXA2. By day three of differentiation, the endoderm cells have differentiated into foregut progenitors that appear polygonal in shape.Later, by day six, the foregut progenitors have differentiated into liver bud progenitors. These liver bud progenitors express AFP, TPX3, and HNF4alpha. Across three hPSC lines, this method generates day six AFP-positive liver progenitors at an efficiency of approximately 89%Finally, after 18 days of liver differentiation from hPSCs, albumin-positive hepatocyte-like cells appear.Morphologically, these cells appear epithelial, forming bright borders reminiscent of bile canaliculi. At this stage, the cytoplasm of the day 18 hPSC-derived hepatocytes appears darker than the nucleus. When seeding human pluripotent stem cells for differentiation, it is imperative to seed them at the right density and also to distribute them across the well by shaking the plate.The ability to produce human hepatocytes in vitro is an enabling technology that will allow scientists to investigate mechanisms underlying liver development. This method produces large numbers of human hepatocytes in vitro which could be used to investigate liver function and disease and to eventually enable new therapies for liver failure.

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