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.

efficient-differentiation-human-pluripotent-stem-cells-into-liver

Research

JoVE Journal

Developmental Biology

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of various trophoblastic cells that differentiate from the extra-embryonic trophectoderm cells of the preimplantation blastocyst. As such, our understanding of the early differentiation events of the human placenta is limited because of ethical and legal restrictions on the isolation and manipulation of human embryogenesis. Human pluripotent stem cells (hPSCs) are a robust model system for investigating human development and can also be differentiated in vitro into trophoblastic cells that express markers of the various trophoblast cell types. Here, we present a detailed protocol for differentiating hPSCs into trophoblastic cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal signaling pathways. This protocol generates various trophoblast cell types that can be transfected with siRNAs for investigating loss-of-function phenotypes or can be infected with pathogens. Additionally, hPSCs can be genetically modified and then differentiated into trophoblast progenitors for gain-of-function analyses. This in vitro differentiation method for generating human trophoblasts starting from hPSCs overcomes the ethical and legal restrictions of working with early human embryos, and this system can be used for a variety of applications, including drug discovery and stem cell research.

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

Here, we present a protocol to efficiently generate human trophoblastic cells from human pluripotent stem cells using bone morphogenic protein 4 and inhibitors of the Activin/Nodal pathways. This method is suitable for the efficient differentiation of human pluripotent stem cells and can generate large quantities of cells for genetic manipulation.

The placenta is the first organ to develop during embryogenesis and is required for the survival of the developing embryo. The placenta is comprised of ...

Protocol for the Differentiation of Human Induced Pluripotent Stem Cells into Mixed Cultures of Neurons and Glia for Neurotoxicity Testing

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is that the reprogramming of somatic cells to produce human induced pluripotent stem cells (hiPSCs) avoids the ethical and legislative issues related to the use of human embryonic stem cells (hESCs). HiPSCs can be expanded and efficiently differentiated into different types of neuronal and glial cells, serving as test systems for toxicity testing and, in particular, for the assessment of different pathways involved in neurotoxicity. This work describes a protocol for the differentiation of hiPSCs into mixed cultures of neuronal and glial cells. The signaling pathways that are regulated and/or activated by neuronal differentiation are defined. This information is critical to the application of the cell model to the new toxicity testing paradigm, in which chemicals are assessed based on their ability to perturb biological pathways. As a proof of concept, rotenone, an inhibitor of mitochondrial respiratory complex I, was used to assess the activation of the Nrf2 signaling pathway, a key regulator of the antioxidant-response-element-(ARE)-driven cellular defense mechanism against oxidative stress.

Human induced pluripotent stem cells (hiPSCs) are considered a powerful tool for drug and chemical screening and for the development of new in vitro models for toxicity testing, including neurotoxicity. Here, a detailed protocol for the differentiation of hiPSCs into neurons and glia is described.

Human pluripotent stem cells can differentiate into various cell types that can be applied to human-based in vitro toxicity assays. One major advantage is ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

Rapid, Directed Differentiation of Retinal Pigment Epithelial Cells from Human Embryonic or Induced Pluripotent Stem Cells

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing a detailed and thorough protocol is to clearly demonstrate each step and to make this readily available to researchers in the field. This protocol results in a homogenous layer of RPE with minimal or no manual dissection needed. The method presented here has been shown to be effective for induced pluripotent stem cells (iPSC) and human embryonic stem cells. Additionally, we describe methods for cryopreservation of intermediate cell banks that allow long-term storage. RPE generated using this protocol might be useful for iPSC disease-in-a-dish modeling or clinical application.

This protocol describes how to produce retinal pigment epithelial cells (RPE) from pluripotent stem cells. The method uses a combination of growth factors and small molecules to direct the differentiation of stem cells into immature RPE in fourteen days and mature, functional RPE after three months.

We describe a robust method to direct the differentiation of pluripotent stem cells into retinal pigment epithelial cells (RPE). The purpose of providing ...

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

RNA-based Reprogramming of Human Primary Fibroblasts into Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative therapeutic requires a safe, robust, and expedient reprogramming protocol. Here, we present a clinically relevant, step-by-step protocol for the extremely high-efficiency reprogramming of human dermal fibroblasts into iPSCs using a non-integrating approach. The core of the protocol consists of expressing pluripotency factors (SOX2, KLF4, cMYC, LIN28A, NANOG, OCT4-MyoD fusion) from in vitro transcribed messenger RNAs synthesized with modified nucleotides (modified mRNAs). The reprogramming modified mRNAs are transfected into primary fibroblasts every 48 h together with mature embryonic stem cell-specific microRNA-367/302 mimics for two weeks. The resulting iPSC colonies can then be isolated and directly expanded in feeder-free conditions. To maximize efficiency and consistency of our reprogramming protocol across fibroblast samples, we have optimized various parameters including the RNA transfection regimen, timing of transfections, culture conditions, and seeding densities. Importantly, our method generates high-quality iPSCs from most fibroblast sources, including difficult-to-reprogram diseased, aged, and/or senescent samples.

Here we describe a clinically relevant, high-efficiency, feeder-free method to reprogram human primary fibroblasts into induced pluripotent stem cells using modified mRNAs encoding reprogramming factors and mature microRNA-367/302 mimics. Also included are methods to assess reprogramming efficiency, expand clonal iPSC colonies, and confirm expression of the pluripotency marker TRA-1-60.

Induced pluripotent stem cells (iPSCs) have proven to be a valuable tool to study human development and disease. Further advancing iPSCs as a regenerative ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Transient Treatment of Human Pluripotent Stem Cells with DMSO to Promote Differentiation

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and iPSCs) across various lineages remain. Numerous differentiation protocols have been developed, yet variability across cell lines and low rates of differentiation impart challenges in successfully implementing these protocols. Described here is an easy and inexpensive means to enhance the differentiation capacity of PSCs. It has been previously shown that treatment of stem cells with a low concentration of dimethyl sulfoxide (DMSO) significantly increases the propensity of a variety of PSCs to differentiate to different cell types following directed differentiation. This technique has now been shown to be effective across different species (e.g., mouse, primate, and human) into multiple lineages, ranging from neurons and cortical spheroids to smooth muscle cells and hepatocytes. The DMSO pretreatment improves PSC differentiation by regulating the cell cycle and priming stem cells to be more responsive to differentiation signals. Provided here is the detailed methodology for using this simple tool as a reproducible and widely applicable means to more efficiently differentiate PSCs to any lineage of choice.

Generating differentiated cell types from human pluripotent stem cells (hPSCs) holds great therapeutic promise but remains challenging. PSCs often exhibit an inherent inability to differentiate even when stimulated with a proper set of signals. Described here is a simple tool to enhance multilineage differentiation across a variety of PSC lines.

Despite the growing use of pluripotent stem cells (PSCs), challenges in efficiently differentiating embryonic and induced pluripotent stem cells (ESCs and ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...