This study was supported with awards from the UK Regenerative Medicine Platform (MRC MR/L022974/1)&#160;and the Chief Scientist&#8217;s Office (TCS/16/37).

1. Preparation of Agarose Microplate Molds
NOTE: Media used for these experiments must be sterile and at room temperature (RT) for cell culture.
<ol>
	<li>Prepare 2% agarose molds.
	<ol>
		<li>Dissolve 2 g of low melting temperature agarose into 100 mL of sterilized distilled water. Carefully heat in a microwave with interval shaking to dissolve completely.</li>
		<li>Add 520 &#956;L of melted agarose to a 256-well format mold and leave to solidify.</li>
		<li>Transfer each agarose microplate into a single well of a 12-well plate.</li>
		<li>Add 1.5 mL of 1x Dulbecco&#8217;s phosphate-buffered saline (DPBS) with Ca2+/Mg2+ to each well and remove the air bubbles from the microwells by gently pipetting up and down several times using a P1000 pipette tip. This is performed to ensure uniform cell seeding. 
		NOTE: Agarose microplates can be stored for up to 6 months at 4 &#176;C in 1x DPBS.</li>
	</ol>
	</li>
</ol>
2. Seeding Human Pluripotent Stem Cells into Agarose Microwell Plates
<ol>
	<li>Preparation of cell suspension
	<ol>
		<li>Aspirate the medium from an undifferentiated culture of hPSCs as previously described8. 
		NOTE: hPSCs are cultured on LN-521 in mTeSR1 with the medium changed every 24 h, and passaged regularly once the cells reach 75% to 85% of confluency as previously described8.</li>
		<li>Rinse the cells with 5 mL of RT 1x DPBS without Ca2+/Mg2+ and remove the buffer.</li>
		<li>Add 5 mL of 1x cell dissociation reagent (Table of Materials) to the cells and allow cell dissociation by incubating cells at 37 &#176;C for 6-8 min. 
		NOTE: To stop the reaction, examine cell detachment under the microscope. The cells should be partially detached from the plate. Extend the incubation for an extra 1-2 min if longer time is required.</li>
		<li>Stop the reaction by removing the cell dissociation reagent and add 5 mL of fresh mTeSR1 medium supplemented with 10 &#956;M Rho-associated kinase (ROCK) inhibitor Y27632 to the cells. Dissociate cells by pipetting up and down several times by using a P1000 pipette tip.</li>
		<li>Count the viable cells using a hemocytometer and trypan blue staining exclusion. Following this, prepare the cell suspension at the required concentration.</li>
		<li>Calculate the total number of cells needed. For 3D hepatosphere differentiation, seed 3.84 x 105 cells per agarose microplate to generate spheroids with 100-150 &#956;m in diameter.</li>
		<li>Transfer the desired number of cells into a sterile 15 mL or 50 mL centrifuge tube and centrifuge the tube at 200 x g for 5 min at RT to pellet the cells.</li>
		<li>Aspirate and discard the supernatant and resuspend cells in mTeSR1 medium plus 10 &#956;M ROCK inhibitor Y27632. Dilute the cell pellet in the appropriate volume of medium to a final concentration of 2.1 x 106 cells/mL.</li>
	</ol>
	</li>
	<li>Seeding the cells to the prepared agarose microplates 
	NOTE: If using fridge-stored agarose microplates, place them in the cell incubator at 37 &#176;C for at least an hour prior to use and aspirate the 1x DPBS from the well before cell seeding.
	<ol>
		<li>Add 190 &#956;L of the cell suspension into the agarose microwell.</li>
		<li>After seeding, return the plates to the cell incubator at 37 &#176;C and 5% CO2 for 2 h to allow the cells to settle.</li>
		<li>After 2 h, add 1 mL of fresh and warm mTeSR1 medium supplemented with 10 &#956;M ROCK inhibitor Y27632 to each well.</li>
		<li>Return the plates to the cell incubator at 37 &#176;C and 5% CO2 for 24 h and examine the formation of spheres the next day to allow the cells to attach.</li>
	</ol>
	</li>
</ol>
3. Differentiating hPSCs to 3D Hepatospheres on Agarose Microwells
<ol>
	<li>Preparation of poly 2-hydroxyethyl methacrylate (poly-HEMA) coated wells
	<ol>
		<li>Dissolve 2 g of poly-HEMA in 100 mL of 95% ethanol. Stir the solution overnight using a hot plate at 55 &#176;C. Add 250 &#956;L of poly-HEMA solution per well of a 24-well plate and dry overnight at 60 &#176;C using an oven.</li>
	</ol>
	</li>
	<li>Preparation of the differentiation medium
	<ol>
		<li>Prepare a 1,000x stock solution of human activin A by dissolving human activin A lyophilized protein in sterile 0.2% bovine serum albumin (BSA)/DPBS to a final concentration of 100 &#956;g/mL. Store at -20 &#176;C in small aliquots. Use at 1:1,000.</li>
		<li>Prepare a 1,000x stock solution of Wnt3a by dissolving mouse Wnt3a lyophilized protein in sterile 0.2% BSA/DPBS to a final concentration of 10 &#956;g/mL. Store at -20 &#176;C in small aliquots. Use at 1:200.</li>
		<li>Prepare a 1,000x stock solution of human hepatocyte growth factor (HGF) by dissolving human HGF lyophilized protein in sterile 0.2% BSA/DPBS to a final concentration of 10 &#956;g/mL. Store at -20 &#176;C in small aliquots. Use at 1:1,000.</li>
		<li>Prepare a 1,000x stock solution of oncostatin M (OSM) by dissolving OSM in sterile 0.2% BSA/DPBS to a final concentration of 20 &#956;g/mL. Store at -20 &#176;C in small aliquots. Use at 1:1,000.</li>
		<li>Prepare a 1000x stock solution of epithelial growth factor (EGF) by dissolving the lyophilized protein in sterile 0.2% BSA/DPBS to a final concentration of 10 &#956;g/mL. Store at -20 &#176;C in small aliquots. Use at 1:1,000.</li>
		<li>Prepare a 1,000x stock solution of basic fibroblast growth factor (bFGF) by dissolving the lyophilized protein in sterile 0.2% BSA/DPBS to a final concentration of 10 &#956;g/mL. Store at -20 &#176;C in small aliquots. Use at 1:1,000.</li>
		<li>Prepare a 1,000x stock solution of vascular endothelial growth factor (VEGF) by dissolving the lyophilized protein in 0.2% BSA/DPBS to a final concentration of 10 &#956;g/mL. Store at -20 &#176;C in small aliquots. Use at 1:1,000.</li>
		<li>Make endoderm differentiation medium consisting of Roswell Park Memorial Institute 1640 (RPMI 1640) basal medium supplemented with 2% B27 supplement (50x, without insulin), and 1% penicillin/streptomycin (final concentrations: 100 IU/mL and 100 &#956;g/mL, respectively). Unless indicated, at each medium change, supplement the required volume with Wnt3a and activin A at a final concentration of 50 ng/mL and 100 ng/mL, respectively. 
		NOTE: Store at 4 &#176;C and use within two weeks.</li>
		<li>Make hepatoblast differentiation medium consisting of knockout Dulbecco&#39;s modified Eagle medium (KO-DMEM) with 20% knockout serum replacement (KOSR) and supplemented with 0.5% of an alternative supplement to L-glutamine, 1% non-essential amino acids (NEAA), 0.1 mM beta-mercaptoethanol, 1% dimethyl sulfoxide (DMSO), and 1% penicillin/streptomycin (final concentrations at 100 IU/mL and 100 &#956;g/mL, respectively). Filter under vacuum. 
		NOTE: Store at 4 &#176;C and use within two weeks.</li>
		<li>Make hepatocyte maturation medium consisting of hepatocyte medium supplemented with 1% of an alternative supplement to L-glutamine, 10 &#956;M hydrocortisone 21-hemisuccinate sodium salt (HCC), 1% penicillin/streptomycin (final concentrations at 100 IU/mL and 100 &#956;g/mL, respectively). For each medium change, supplement the required volume with OSM and HGF (final concentrations at 20 ng/mL and 10 ng/mL, respectively). 
		NOTE: Store the stock at 4 &#176;C and use within two weeks.</li>
		<li>Make hepatocyte maintenance medium consisting of William&#39;s E medium supplemented with 10% knockout serum replacement, consisting of 1% of an alternative supplement to L-glutamine, 1% penicillin/streptomycin (final concentrations at 100 IU/mL and 100 &#956;g/mL, respectively). For each medium change, supplement the required volume with HGF, EGF, bFGF and VEGF (final concentration of each growth factor at 10 ng/mL). 
		NOTE: Store the stock at 4 &#176;C and use within two weeks.</li>
	</ol>
	</li>
	<li>Check hepatosphere formation 24 h post seeding and initiate hepatocyte differentiation. Carefully remove the mTeSR1 medium and replace it with 1 mL of fresh endoderm differentiation medium supplemented with 100 ng/mL activin A and 50 ng/mL Wnt3a.</li>
	<li>Change supplemented endoderm priming medium every 24 h for 3 days for hESCs. When working with hiPSCs, extend this stage for a further 2 days supplementing the media with activin A (100ng/mL) alone.</li>
	<li>Following definitive endoderm induction, switch to hepatoblast differentiation medium for hepatoblast specification for 5 days replacing the medium every 2 days and perform the last change on the last day of the hepatoblast specification.</li>
	<li>Transfer hepatospheres into the poly-HEMA coated wells.
	<ol>
		<li>Wash the cells once with hepatocyte maturation medium without supplements after removing KSR/DMSO medium and add 1 mL of hepatocyte maturation medium supplemented with 10 ng/mL HGF and 20 ng/mL OSM.</li>
		<li>Using a P1000 pipette, lift up the hepatospheres from the agarose microplate by pipetting up and down the solution several times.</li>
		<li>Transfer the medium containing the hepatospheres to a poly-HEMA coated well.</li>
		<li>Wash the agarose microplate using 1 mL of hepatocyte maturation medium supplemented with 10 ng/mL HGF and 20 ng/mL OSM and transfer the medium to the poly-HEMA coated well.</li>
		<li>Repeat step 3.6.4 as many times as possible in order to transfer all the hepatospheres from the agarose microplate. 
		NOTE: It is critical to carefully pipette up and down the solution containing the hepatospheres to avoid damaging them.</li>
	</ol>
	</li>
	<li>Carefully aspirate excess medium without removing hepatospheres by using a P100 pipette until ~1 mL of medium containing the hepatospheres remains in the poly-HEMA coated well.</li>
	<li>Change the medium every 48 h for 12 days.</li>
	<li>At day 20 when working with hESCs or day 22 if working with hiPSCs, switch the medium to hepatocyte maintenance medium.
	<ol>
		<li>Remove hepatocyte maturation medium and wash cells once with hepatocyte maintenance medium without the supplements. Add 1 mL of hepatocyte maintenance medium supplemented with 10 ng/mL of HGF, EGF, FGF and VEGF.</li>
	</ol>
	</li>
	<li>Change the medium for fresh hepatocyte maintenance medium every 48 h.</li>
</ol>
4. Functional Analysis of Long-term Cultured 3D Hepatospheres
<ol>
	<li>Switch to hepatocyte maturation medium supplemented with 10 &#956;M HCC, L-glutamine, 1% penicillin/streptomycin (final concentrations at 100 IU/mL and 100 &#956;g/mL, respectively) and 10 ng/mL HGF 48 h prior to performing functional analysis.</li>
	<li>Analyze hepatocyte metabolic function using cytochrome (CYP) P450 assays.
	<ol>
		<li>Replace medium with 1 mL of fresh hepatocyte maturation medium supplemented with 50 &#956;M luciferin-6&#39;-pentafluoro-benzyl ether (luciferin-PFBE) substrate to detect CYP3A basal activity or 100 &#956;M luciferin-methyl ether (luciferin-ME) substrate to detect CYP1A2 basal activity (number of replicates = 3). Use tissue culture media as a negative control. 
		NOTE: In order to avoid cross-reactivity, it is recommended not to use the same wells containing 3D hepatospheres to test different CYP P450 activities but to perform them in individual wells.</li>
		<li>Incubate cells for 24 h at 37 &#176;&#160;C.</li>
		<li>Collect the supernatants using a P100 pipette tip and carry out the assay as per manufacturer&#8217;s instructions.</li>
		<li>Measure the relative levels of basal activity and normalize to per mg protein as determined by the bicinchoninic acid assay (BCA).</li>
	</ol>
	</li>
	<li>Test serum protein production using enzyme-linked immunosorbent assay (ELISA).
	<ol>
		<li>Replace medium with 1 mL of fresh hepatocyte maturation medium supplemented with 10 ng/mL HGF and 20 ng/mL OSM (number of replicates = 3). Use tissue culture media as a negative control.</li>
		<li>Incubate cells for 24 h at 37&#160;&#176; C.</li>
		<li>Collect the supernatant using a P100 pipette tip and measure the relative levels of serum protein production as per manufacturer&#39;s instructions.</li>
		<li>Normalize to per mg protein as determined by the BCA Assay.</li>
	</ol>
	</li>
</ol>
5. Immunocytochemistry
<ol>
	<li>Preparation of paraffin sections containing hepatospheres.
	<ol>
		<li>Wash the hepatospheres three times with 1x DPBS.</li>
		<li>Fix the hepatospheres with ice-cold methanol for 30 min.</li>
		<li>Wash three times with 1x DPBS.</li>
		<li>Embed the hepatospheres in 300 &#956;L of a tempered solution of 2% agarose dissolved in H2O using an empty well of a 24-well plate as a mold and leave it to solidify for 30 min.</li>
		<li>Embed the agarose containing hepatospheres in paraffin.</li>
		<li>Section the paraffin block containing fixed hepatospheres in 4 &#956;m thick sections using a microtome.</li>
	</ol>
	</li>
	<li>De-waxing and rehydration of the sections. 
	NOTE: Use fresh solutions whenever it is possible. Do not use solutions that have been on the staining trough for longer than a week.
	<ol>
		<li>Place sections in a slide rack.</li>
		<li>Immerse slide rack containing sections in a staining trough containing 300 mL of xylene for 5 min.</li>
		<li>Repeat step 5.2.2.</li>
		<li>Immerse in absolute ethanol for 20 s.</li>
		<li>Immerse in 95% ethanol for 20 s.</li>
		<li>Immerse in 90% ethanol for 20 s.</li>
		<li>Immerse in 80% ethanol for 20 s.</li>
		<li>Immerse in 70% ethanol for 20 s.</li>
		<li>Rehydrate the sections with water for 5 min. 
		NOTE: Maintain the slides in the same slide rack and move it from one staining trough to the next one. Volume used (generally ~300 mL) depends on the size of the staining trough.</li>
	</ol>
	</li>
	<li>Antigen retrieval of paraffin sections containing hepatospheres.
	<ol>
		<li>Heat de-waxed and rehydrated sections in 1x Tris-EDTA (TE) pH 9.0 buffer solution for 15 min in a microwave at 800 W.</li>
		<li>Cool down samples by immersing them in tap water for 5 min.</li>
	</ol>
	</li>
	<li>Immunostaining.
	<ol>
		<li>Incubate slides with blocking solution made of PBS with 0.1% polysorbate 20 (PBS/T)/10% BSA for 1 h at RT.</li>
		<li>Replace blocking solution with the appropriate primary antibody diluted in PBS/T/1% BSA and incubate at 4 &#176;C with gentle agitation overnight.</li>
		<li>Wash cells with PBS/T for 5 min and repeat three times.</li>
		<li>Incubate with the appropriate secondary antibody diluted in PBS/T/1% BSA and incubate in the dark at RT for 1 h with gentle agitation. 
		NOTE: The optimized primary and secondary antibodies are listed in Table of Materials.</li>
		<li>Wash cells with PBS/T for 5 min and repeat three times.</li>
		<li>Add 4&#39;,6-diamidino-2-phenylindole (DAPI) to the cells according to manufacturer&#8217;s instructions and place a glass coverslip gently to reduce air bubbles. Keep fixed cells at 4 &#176;C in the dark. Observe staining under a microscope with appropriate filter and fluorescent lamp. 
		NOTE: Store plates at 4 &#176;C in the dark until imaging.</li>
	</ol>
	</li>
</ol>

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David C. Hay is a co-founder and shareholder of Stemnovate Ltd and HigherSteaks Ltd. The rest of the authors certify that they have no conflicts of interest in the subject matter or materials discussed in this article.

The development of defined and xeno-free systems to produce human hepatospheres in 3D is required for both in vitro and in vivo endeavors. At present most hepatocyte differentiation approaches from human pluripotent stem cells are performed in two dimensional adherent cultures. These environments lack many of the environmental cues involved in tissue genesis and homeostasis which include; heterotypic cell interactions, matrix production and remodeling, resulting in poor translation to the in vivo biology18,19.
As a result, research has focused on alternative approaches to generate hepatospheres from pluripotent stem cells. A number of 3D studies have advanced the field, but those are reliant on animal products20,22,24 to provide support and/or require the use of human tissue21,22 which complicates technology scale-up and compromises experimental reproducibility and application.
The procedure described in our article (Figure 1) is defined, efficient, highly reproducible and cost-effective, allowing the production of functional liver spheres, which remain functional over a year in vitro and provide critical liver support in vivo14. Importantly, this platform allows the user to control the size of the 3D liver spheres, limiting the formation of dense necrotic centers and loss of phenotype.
The transfer of the 3D hepatospheres to the poly-HEMA coated plates represents a critical step in this protocol. It is important to pipette gently at this stage in the procedure to avoid damaging the sphere. In addition, media changes must be performed carefully to avoid shear stress and distortion of sphere structure.
In these studies, 3D hepatospheres displayed an organized structure (Figure 2 and Figure 3), cytochrome P450 function (Figure 4) and secreted liver proteins, including albumin and alpha-fetoprotein (Figure 5). This procedure has been successfully performed in four pluripotent stem cells lines with comparable outcomes. Looking ahead, this technology could be employed as a platform to develop further endodermal and mesenchymal tissues with complex architectures.

The ability of human pluripotent stem cells (hPSCs) to self-renew, whilst retaining pluripotency, provides an opportunity to produce human cell types and tissues on demand. hPSCs have been efficiently differentiated into hepatocyte-like cells (HLCs) using two-dimensional (2D) adherent culture systems1,2,3,4,5,6,7,8,9,10. These systems have been used to successfully model monogenic disease, virus lifecycle, drug induced liver injury (DILI), fetal exposure to toxins and non-alcoholic fatty liver disease (NAFLD)11,12,13,14,15. However, these models do possess some drawbacks, which limit their routine use. Those include fetal marker expression, unstable phenotype and poor tissue architecture16,17,18,19, which could also limit extrapolation to organ function in vivo.
To overcome these limitations, three-dimensional (3D) differentiation platforms have been developed to mimic in vivo tissue architecture. Although enabling, those approaches rely on the use of animal derived products and matrices to drive tissue genesis20,21,22, limiting scale-up and widespread application.
Here, we detail procedures to generate large quantities of 3D hepatospheres from hPSCs using defined materials and cell self-assembly. Notably, the tissue generated by our procedure remains functional for more than one year in cell culture and is capable of supporting liver function in vivo23.
In summary, our defined differentiation approach allows the generation of stable human hepatospheres from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). We believe the described procedure represents a significant breakthrough in the generation of 3D hepatospheres for basic and applied scientific research.

<table><tbody><tr><td>&lt;strong&gt;Cell Culture and functional assays&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose</td><td>Fisher Bioreagents</td><td>10766834</td><td></td></tr><tr><td>B27 supplement&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>12587-010</td><td></td></tr><tr><td>beta-mercaptoethanol</td><td>Life Technologies&amp;nbsp;</td><td>31350</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A2058&amp;nbsp;</td><td></td></tr><tr><td>DAPI&amp;nbsp;</td><td>Invitrogen</td><td>D1306</td><td></td></tr><tr><td>DMSO&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D5879</td><td></td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>ThermoFisher&amp;nbsp;</td><td>14190250</td><td></td></tr><tr><td>DPBS with Calcium and Magnesium</td><td>ThermoFisher&amp;nbsp;</td><td>14040133</td><td></td></tr><tr><td>Gentle cell dissociation reagent</td><td>STEMCELL Technologies&amp;nbsp;</td><td>7174</td><td></td></tr><tr><td>GlutaMax&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>35050</td><td></td></tr><tr><td>HepatoZYME-SFM</td><td>Life Technologies&amp;nbsp;</td><td>&amp;nbsp; 17705021</td><td></td></tr><tr><td>Human Activin A&amp;nbsp;</td><td>Peprotech&amp;nbsp;</td><td>120-14E</td><td></td></tr><tr><td>Human Alpha Fetoprotein ELISA</td><td>Alpha Diagnostics</td><td>500</td><td></td></tr><tr><td>Human Basic Fibrobaslt Growth Factor</td><td>Peprotech&amp;nbsp;</td><td>100-18B</td><td></td></tr><tr><td>Human Epithelial Gropwth Factor</td><td>Peprotech&amp;nbsp;</td><td>236-EG</td><td></td></tr><tr><td>Human Hepatocyte Growth Factor</td><td>Peprotech&amp;nbsp;</td><td>100-39</td><td></td></tr><tr><td>Human Oncostatin M&amp;nbsp;</td><td>Peprotech&amp;nbsp;</td><td>300-10</td><td></td></tr><tr><td>Human Recombinant Laminin 521&amp;nbsp;</td><td>BioLamina&amp;nbsp;</td><td>LN521-02</td><td></td></tr><tr><td>Human Serum Albumin ELISA</td><td>Alpha Diagnostics</td><td>1190</td><td></td></tr><tr><td>Human Vascular Growth Factor</td><td>Bio-techne</td><td>293-VE</td><td></td></tr><tr><td>Hydrocortisone 21-hemisuccinate sodium salt</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H4881</td><td></td></tr><tr><td>Knockout DMEM&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>10829</td><td></td></tr><tr><td>Knockout Serum Replacement&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>10828</td><td></td></tr><tr><td>Micro-mold spheroids&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>Z764000</td><td></td></tr><tr><td>mTeSR1 medium&amp;nbsp;</td><td>STEMCELL Technologies&amp;nbsp;</td><td>5850</td><td></td></tr><tr><td>Non-essential amino acids&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>11140</td><td></td></tr><tr><td>P450-Glo CYP1A2 Assay and Screening System</td><td>Promega</td><td>V8771</td><td></td></tr><tr><td>P450-Glo CYP3A4 Assay and Screening System</td><td>Promega</td><td>V8801</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Life Technologies&amp;nbsp;</td><td>15140122</td><td></td></tr><tr><td>poly-HEMA (Poly 2-hydorxyethyl methacrylate)&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P3932</td><td></td></tr><tr><td>Recombinant mouse Wnt3a&amp;nbsp;</td><td>Bio-techne</td><td>1324-WN-500/CF</td><td></td></tr><tr><td>RPMI 1640&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>21875</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Microwave</td><td>Bosch</td><td></td><td></td></tr><tr><td>Microtome</td><td>Leika</td><td>RM2125RT</td><td></td></tr><tr><td>Oven</td><td>Thermoscientific</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Albumin&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>A6684&amp;nbsp;</td><td>1:100 (mouse)</td></tr><tr><td>CYP3A4</td><td>University of Dundee</td><td>University of Dundee</td><td>1:200 (sheep)</td></tr><tr><td>E-cadherin</td><td>Abcam</td><td>ab76055</td><td>1:200 (mouse)</td></tr><tr><td>HNF-4&amp;alpha;</td><td>Santa Cruz</td><td>sc-8987</td><td>1:100 (rabbit)</td></tr><tr><td>IgG</td><td>DAKO</td><td>X0943</td><td>1:400</td></tr><tr><td>Vimentin</td><td>DAKO</td><td>M0725</td><td>1:100 (sheep)</td></tr></tbody></table>

serum free production of three-dimensional human hepatospheres from pluripotent stem cells

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) represent promising sources of human liver spheres. We have developed a serum free and defined method of cellular differentiation to generate three-dimensional human liver spheres formed from human pluripotent stem cells. A potential limitation of the technology is the production of dense spheres with dead material inside. In order to circumvent this, we have employed agarose microwell technology at defined cell densities to control the size of the 3D spheres, preventing the generation of apoptotic and/or necrotic cores.&#160; Notably, the spheres generated by our approach display liver function and stable phenotype, representing a valuable resource for basic and applied scientific research. We believe that our approach could be used as a platform technology to develop further tissues to model and treat human disease and in the future may permit the generation of human tissue with complex tissue architecture.

Three-dimensional aggregates from the embryonic stem cell line (H9) or the induced pluripotent stem cell line (P106) were differentiated towards the hepatocyte lineage using our defined procedure (Figure 1). Pluripotent stem cells were first primed towards definitive endoderm prior to hepatoblast specification. Following this, hepatoblasts were matured into 3D hepatospheres which could be maintained in culture for up to one year23.
To study the structure of 3D spheres, 30 day-old hESC- or iPSC-derived spheres were fixed, sectioned and stained to detect the presence of proteins expressed in hepatocytes and mesenchymal cells. Hepatocyte nuclear factor 4 alpha (HNF4&#945;) and the mesenchymal marker vimentin were employed, revealing the presence of an outer layer composed of hepatocyte like cells surrounding a core of mesenchymal cells (Figure 2). We followed these experiments analyzing&#160;the expression of proteins expressed in hepatocytes: albumin, CYP3A and E-cadherin. Immunostaining revealed that the expression of these proteins was restricted to the outer layer of the spheres (Figure 3).
Functional analyses of the hepatospheres were performed in day 30 cultures. CYP1A2 and CYP3A are important enzymes within functional hepatocytes. Their activity was assessed using established assays. H9-derived hepatospheres exhibited CYP3A activity of&#160;220,375 &#177; 74514 RLU/mL/mg protein and CYP1A2 activity of&#160;732,440 &#177; 33,330 RLU/mL/mg protein (Figure 4A). CYP3A activity in P106-derived hepatospheres was 132117 &#177; 43,391 RLU/mL/mg protein and CYP1A2 activity was&#160;409,907 &#177; 121,723 RLU/mL/mg protein (Figure 4B). When compared with two batches of human primary hepatocytes, 3D liver spheres displayed respectable levels of CYP activity10.
Analysis of the synthesis and secretion of albumin and alpha-fetoprotein (AFP) revealed that H9-derived hepatospheres secreted 683.9 &#177; 84 and 159 &#177; 20 ng/mL/24 h/mg protein of albumin and alpha-fetoprotein, respectively (Figure 5A). Whereas P106-derived hepatospheres secreted 497 &#177; 41 and 756 &#177; 24 ng/mL/24 h/mg protein of albumin and alpha-fetoprotein, respectively (Figure 5B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59965/59965fig1.jpg" /> 
Figure 1: Stepwise differentiation procedure to generate 3D hepatospheres from hESCs. Blue brackets represent days of differentiation for hiPSCs. <a href="https://www.jove.com/files/ftp_upload/59965/59965fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59965/59965fig2.jpg" /> 
Figure 2: Structural reorganization of 3D hepatospheres. Representative images of the expression of hepatocyte nuclear factor 4 alpha (HNF4&#945; - green) and vimentin (red) in (A) H9-derived 3D hepatospheres and (B) P106-derived 3D hepatospheres and their corresponding immunoglobulin G (IgG) controls. Scale bars represent 60 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59965/59965fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59965/59965fig3.jpg" /> 
Figure 3: Evaluation of hepatic marker expression in 3D hepatospheres. Representative images of the expression of hepatocyte markers -&#160;albumin, CYP3A, E-cadherin and their corresponding IgG controls in (A) H9- and (B) P106-derived 3D hepatospheres. Scale bars = 60 &#956;m. <a href="https://www.jove.com/files/ftp_upload/59965/59965fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59965/59965fig4.jpg" /> 
Figure 4: Cytochrome P450 function in 3D hepatospheres. Measurement of the cytochrome P450 1A2 and 3A activity in (A) H9-derived 3D hepatospheres and (B) P106-derived 3D hepatospheres. The data represents the mean of three biological replicates, and the error bars represent the standard deviation (SD). Activity is quoted as relative light units (RLU) per mL per mg of protein. <a href="https://www.jove.com/files/ftp_upload/59965/59965fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59965/59965fig5.jpg" /> 
Figure 5: Analysis of hepatosphere protein secretion. The secretion of albumin and alpha-fetoprotein (AFP) was analyzed in (A) H9-derived 3D hepatospheres and (B) P106-derived 3D hepatospheres. The data is representative of three biological replicates, and the error bars represent the SD. Secreted protein is quoted as nanograms of protein per mL per 24 h per mg of protein. <a href="https://www.jove.com/files/ftp_upload/59965/59965fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells at JoVE.com

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

This protocol describes an approach to produce hepatospheres from human pluripotent stem cells using a defined culture system and cell self-assembly. This protocol is reproducible in a number of cell lines, cost effective and allows the production of stable human hepatospheres for biomedical application.

The development of renewable sources of liver tissue is required to improve cell-based modelling, and develop human tissue&#160;for transplantation. Human ...

serum-free-production-three-dimensional-human-hepatospheres-from

Research

JoVE Journal

Developmental Biology

8.9K Views.  University of Edinburgh. Deze methogenetica grote hoeveelheid 3D hepatosferen van menselijke pluripotente stamcellen met behulp van de fijne materialen en cellen van de assemblage. De belangrijkste vooruitgang van deze techniek is dat het mogelijk maakt het beheersen van de grootte van de 3D-lever bollen, het beperken van de vorming van necrotische centra en verlies van fenotype. Het aantonen van de procedure met mij zal Yu Wang, een post-doctoraat in ons laboratorium.Bereid je voor om agarose mallen te presen...

Video: Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

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

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

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

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

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

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

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

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

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

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

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

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

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

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

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

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

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

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

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

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

Biology

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

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

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

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

Developing HiPSC Derived Serum Free Embryoid Bodies for the Interrogation of 3-D Stem Cell Cultures Using Physiologically Relevant Assays

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not represent the underlying cytoarchitectural and functional complexity of the affected individuals carrying suspected disease variants. Conventional 2-D models remain incomplete representations of in vivo-like structures and do not adequately capture the complexity of the brain. Thus, there is an emerging need for more 3-D hiPSC-based models that can better recapitulate the cellular interactions and functions seen in an in vivo system.
Here we report a protocol to develop a 3-D system from undifferentiated hiPSCs based on the serum free embryoid body (SFEB). This 3-D model mirrors aspects of a developing ventralized neocortex and allows for studies into functions integral to living neural cells and intact tissue such as migration, connectivity, communication, and maturation. Specifically, we demonstrate that the SFEBs using our protocol can be interrogated using physiologically relevant and high-content cell based assays such as calcium imaging, and multi-electrode array (MEA) recordings without cryosectioning. In the case of MEA recordings, we demonstrate that SFEBs increase both spike activity and network-level bursting activity during long-term culturing. This SFEB protocol provides a robust and scalable system for the study of developing network formation in a 3-D model that captures aspects of early cortical development.

Here we report a protocol to develop a three-dimensional (3-D) system from human induced-pluripotent stem cells (hiPSCs) called the serum free embryoid body (SFEB). This 3-D model can be used like an organotypic slice culture to model human cortical development and for the physiological interrogation of developing neural circuits.

Although a number of in vitro disease models have been developed using hiPSCs, one limitation is that these two-dimensional (2-D) systems may not ...

Neuroscience

Human Liver Spheroids from Peripheral Blood for Liver Disease Studies

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to their nature to cluster in the culture dishes with low or no adhesive characteristics, they form aggregates of multiple liver cells that are called human liver spheroids. The forming of 3D liver spheroids relies on the natural tendency of hepatic cells to aggregate in the absence of an adhesive substrate. These 3D structures possess better physiological responses than cells, which are closer to an in vivo environment. Using 3D hepatocyte cultures has numerous advantages when compared with classical two-dimensional (2D) cultures, including a more biologically relevant microenvironment, architectural morphology that reassembles natural organs as well as a better prediction regarding disease state and in vivo-like responses to drugs. Various sources can be used to generate spheroids, like primary liver tissue or immortalized cell lines. The 3D liver tissue can also be engineered by using human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs) to derive hepatocytes. We have obtained human liver spheroids using blood-derived pluripotent stem cells (BD-PSCs) generated from unmanipulated peripheral blood by activation of human membrane-bound GPI-linked protein and differentiated to human hepatocytes. The BD-PSCs-derived human liver cells and human liver spheroids were analyzed by light microscopy and immunophenotyping using human hepatocyte markers.

Here we present a non-genetic method to generate human autologous liver spheroids using mononuclear cells isolated from steady-state peripheral blood.

Human liver cells can form a three-dimensional (3D) structure capable of growing in culture for some weeks, preserving their functional capacity. Due to ...

Bioengineering

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

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

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

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

Immunology and Infection

Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to shortages in donor organ availability. Organ scarcity also affects the routine supply of human hepatocytes for basic research and the clinic. Therefore, the development of renewable sources of human liver progenitor cells is desirable and is the goal of this study. To be able to effectively generate and deploy human liver progenitors on a large scale, a reproducible hepatic progenitor differentiation system was developed. This protocol aids experimental reproducibility between users in a range of cell cultureware formats and permits differentiations using both, human embryonic and induced pluripotent stem cell lines. These are important advantages over current differentiation systems that will enhance the basic research and may pave the way towards clinical product development.

The goal of this article is to provide a standardized approach to induce human hepatic progenitor differentiation from pluripotent stem cells. The development of this procedure with ready-to-use media formulations offer the user a facile system to generate human liver cells for biomedical research and translation.

Liver disease is an escalating global health issue. While liver transplantation is an effective mode of therapy, patient mortality has increased due to ...

Serum Free Production of Three-dimensional Human Hepatospheres from Pluripotent Stem Cells

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