This study was supported with awards from the MRC Doctoral Training Partnership (MR/K501293/1), the UK Regenerative Medicine Platform (MRC MR/L022974/1 and MR/K026666/1), the Chief Scientist Office (TCS/16/37).

1. Human pluripotent stem cell (hPSC) maintenance on laminin-521
<ol>
	<li>Maintain human pluripotent cells (hPSCs) at 37 &#176;C and 5% CO2 in a 6 well plate on laminin-521 (LN-521). Feed the cells daily with 2 mL of stem cell maintenance medium (i.e., mTeSR1 medium) per well of a 6 well plate up to the chosen seeding day for differentiation (day 0).</li>
	<li>Ensure that the desired cell confluency of 70&#8211;80% is achieved prior to cell harvesting.</li>
</ol>
2. Laminin-521 multiwell preparation and hPSC seeding for differentiation
NOTE: For hPSCs not maintained on LN-521 (e.g., matrigel or fibronectin), split hPSCs onto LN-521 and culture for 1 week prior to passaging and eliciting differentiation to improve the efficiency of the process15,17,18.
<ol>
	<li>Laminin-coated plate preparation
	<ol>
		<li>Thaw a vial of recombinant LN-521 (100 &#956;g/mL) at 4 &#176;C for 2 h or overnight.</li>
		<li>Prepare an 8 &#956;g/mL solution by diluting the thawed LN-521 in ice-cold 1x DPBS with Ca2+/Mg2+.</li>
		<li>Add 0.25 mL of the 8 &#956;g/mL LN-521 solution to each well of a 24 well plate or 0.05 mL to each well of a 96 well plate. Rock the plate gently from side to side to evenly coat the wells with the LN-521 solution. 
		NOTE: For the 96 well plate format, volume dispensing, cell seeding, and medium changes can be performed using a semi-automated pipeline. For details see Meseguer-Ripolles et al.19.</li>
		<li>Seal the LN-521 coated plates with a semi-transparent, flexible film and store at 4 &#176;C overnight prior to use. 
		NOTE: LN-521 coated plates can be used for up to 2 weeks when stored at 4 &#176;C. Avoid any drying of the laminin-coated wells.</li>
	</ol>
	</li>
	<li>On the day of the cell seeding, warm up the precoated plates in a cell culture incubator at 37 &#176;C for 30&#8211;60 min.</li>
	<li>Aspirate the LN-521 solution. 
	NOTE: Avoid direct contact of the aspirator with the bottom of the well to prevent damage to the LN-521 coating.</li>
	<li>Dispense 0.5 mL of stem cell maintenance medium with freshly supplemented 10 &#181;M Rho-associated kinase (ROCK) inhibitor Y27632 to each well of a 24 well plate or 0.05 mL to each well of a 96 well plate. Place the plate in the incubator until ready for cell seeding.</li>
	<li>On the scheduled seeding day (day 0) and with an hPSC confluency between 70&#8211;80%, mark any regions of spontaneous differentiation on the bottom of the wells of the 6 well plate. 
	NOTE: Spontaneous differentiation can be visualized by a gross change in the cell size and/or the presence of different cell morphologies using phase contrast microscopy.</li>
	<li>Aspirate and discard the marked regions of differentiation and the spent medium from the wells. Wash each well with 1 mL of DPBS without Ca2+/Mg2+ at room temperature (RT).</li>
	<li>Add 1 mL of enzyme-free dissociation reagent (see Table of Materials) to each well and incubate at 37 &#176;C for 8&#8211;10 min until cells visibly detach from the plate.</li>
	<li>Use a cell scraper to gently detach the cells from the wells. Pipette the contents of each well up and down 2&#8211;4x with a P1000 pipette to yield a single-cell suspension. For each cell line, pool cells from all maintenance wells into a sterile 50 mL tube.</li>
	<li>Wash each emptied well with 1 mL of the stem cell maintenance medium. Add the washes to the corresponding tube containing the pooled cells from the appropriate cell line.</li>
	<li>For each cell line, perform three viable cell counts on the pooled samples. Calculate the average live cell count (live cells/mL) for each cell line.</li>
	<li>Centrifuge the pooled samples at 250 x g for 5 min at RT. Aspirate the supernatant, then resuspend the cell pellet in 1&#8211;3 mL of RT stem cell maintenance medium, freshly supplemented with 10 &#956;M ROCK inhibitor Y27632.</li>
	<li>For each cell line, calculate the cell number needed per the number of wells prepared at step 2.4 (see Table 1). Resuspend the required cell number with stem cell maintenance medium freshly supplemented with 10 &#956;M ROCK inhibitor Y27632.</li>
	<li>Add the calculated volume(s) into the wells of the prepared and precoated plates from step 2.4 without removing the volume added previously. The total volume per well will be 1 mL for a 24 well plate and 0.1 mL for a 96 well plate. Gently rock the plates from side to side and back and forth to ensure even cell dispersion throughout the well. 
	NOTE: An even cell distribution across the well is key to ensure homogenous cell seeding and successful differentiation.</li>
	<li>Place the seeded plates into the incubator and immediately rock the seeded plates gently back and forth and from side to side to evenly distribute the cells and maintain cultures at 37 &#176;C and 5% CO2.</li>
</ol>
3. Differentiating hPSCs to hepatic progenitors on laminin-521
<ol>
	<li>Prepare media for definitive endoderm induction (stage 1) using the Endoderm Basal Medium supplemented with the correct additives.
	<ol>
		<li>On day 0, thaw the bottle of the Endoderm Basal Medium overnight at 4 &#176;C.</li>
		<li>Prepare Stage 1 Medium 1 (for use on day 1) as needed.</li>
		<li>Thaw Supplement MR and Supplement CJ on ice.</li>
		<li>Dilute Supplement MR and Supplement CJ 1:100 in the Endoderm Basal Medium.</li>
		<li>Prepare Stage 1 Medium 2 for use on days 2&#8211;4 as needed.</li>
		<li>Dilute Supplement CJ 1:100 in the Endoderm Basal Medium.</li>
	</ol>
	</li>
	<li>Prepare media for the subsequent hepatic progenitor cell specification (Stage 2) differentiation using the Hepatic Progenitor Medium.
	<ol>
		<li>On day 4, thaw the bottle of the Hepatic Progenitor Medium overnight at 4 &#176;C. 
		NOTE: 1% penicillin/streptomycin (final concentrations of 100 IU/mL and 100 &#956;g/mL, respectively) was used for this experiment. Antibiotics are not required; antibiotic use is at user discretion.</li>
	</ol>
	</li>
	<li>On day 1 of the differentiation, remove the spent stem cell maintenance medium with 10 &#956;M ROCKi Y-27632 medium from the wells and replace with 0.5 mL of complete Stage 1 Medium 1 per well of a 24 well plate and 0.1 mL per well of a 96 well plate.</li>
	<li>On days 2, 3, and 4, remove the spent medium and feed each well with 0.5 mL of Stage 1 Medium 2 per well for a 24 well plate or 0.1 mL per well of a 96 well plate.</li>
	<li>On day 5, fix the wells for definitive endoderm differentiation analysis via immunocytochemistry. For the remaining wells, remove the spent medium and feed each well with 0.5 mL of Hepatic Progenitor Differentiation Medium per well for a 24 well plate or 0.1 mL per well of a 96 well plate. Refresh the medium again on days 6, 7, and 9.</li>
	<li>On day 10, harvest wells for hepatic progenitor differentiation analysis or proceed with further hepatocyte-like cell differentiation. 
	NOTE: At this time point, samples were either fixed with 4% paraformaldehyde (PFA) for immunocytochemistry analysis or supernatant was collected for ELISA and cells were collected for protein quantification.</li>
</ol>
4. Characterization of the hepatic progenitor differentiation cultures generated from hPSCs on laminin-521
<ol>
	<li>On day 5, detect expression of definitive endoderm-specific markers using immunostaining.</li>
	<li>On day 10, detect expression of hepatic progenitor-specific markers using immunostaining.</li>
	<li>On day 10, measure AFP and ALB secretion via ELISA using a kit following manufacturer&#8217;s instructions and normalize per mg protein as determined by a bicinchoninic acid (BCA) protein assay.</li>
	<li>Assess the hepatic progenitor variability of the 96 well plate by quantifying of the percentage of HNF4&#945; positive cells per well.</li>
</ol>
5. Immunocytochemistry and image acquisition
<ol>
	<li>On days 5 and 10 of the differentiation, wash cells 3x with 1x DPBS, with 0.5 mL per well of a 24 well plate and 0.1 mL per well of a 96 well plate. Incubate the plate with gentle shaking for 2&#8211;5 min at RT. 
	NOTE: Use DPSB without Ca2+/Mg2+ for immunocytochemistry.</li>
	<li>Fix the cells with 4% paraformaldehyde (PFA) at RT for 15&#8211;30 min by adding 0.3 mL of PFA per well for a 24 well plate and 0.1 mL per well of a 96 well plate.</li>
	<li>Wash 3x with 1x DPBS&#160;as described in step 5.1.</li>
	<li>Permeabilize the membrane with PBST using 0.1% Tween, 1x DPBS and incubate for 20 min at RT by adding 0.3 mL of PBST per well of a 24 well plate and 0.1 mL per well of a 96 well plate.</li>
	<li>Perform the protein block by incubating the cells with 10% BSA in PBST for 1 h, adding 0.3 mL of BSA per well of a 24 well plate and 0.1 mL of BSA per well of a 96 well plate, gently shaking using a plate shaker.</li>
	<li>After protein blocking, replace the blocking solution with the primary antibody diluted in 1% BSA in PBST and incubate at 4 &#176;C with gentle shaking overnight. 
	NOTE: Do not wash between protein block and antibody addition.</li>
	<li>After 24 h, wash wells 3x with PBST.</li>
	<li>Add the secondary antibody in 1% BSA in PBST. Incubate 1 h at RT in the dark with gentle shaking.</li>
	<li>After secondary antibody incubation, wash wells 3x with 1x DPBS and dispense Hoechst stain according to the manufacturer&#8217;s instructions for 10 min at RT in the dark with gentle shaking.</li>
	<li>Wash 3x with 1x DPBS. The plates are now ready for imaging. 
	NOTE: Store plates at 4 &#176;C in the dark until imaging.</li>
	<li>Image the multiwell plate using a high-content imaging microscope after immunohistochemistry. Image acquisition of several fields of view is recommended to obtain a true representation of the well. The expression of the different markers was assessed via cell segmentation analysis using commercial software (see Table of Materials) (Figure 1). 
	NOTE: Cell segmentation can also be performed using an image analysis open-source software such as CellProfiler or Fiji20,21.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+Human+Pluripotent+Stem+Cells+for+Modeling+Liver+Development+and+Disease.">The Use of Human Pluripotent Stem Cells for Modeling Liver Development and Disease.</a> Hepatology. 69 (3), 1306-1316 (2019).</li><li>Alwahsh, S. M., Rashidi, H., Hay, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+cell+therapy:+is+this+the+end+of+the+beginning.">Liver cell therapy: is this the end of the beginning.</a> Cell and Molecular Life Sciences. 75 (8), 1307-1324 (2018).</li><li>Hay, D. 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David C. Hay is a co-founder and shareholder of Stemnovate 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 generation of human hepatic progenitor cells from pluripotent stem cells on a large scale could represent a promising alternative to cadaver-derived material. Protocol standardization and reproducibility are key to ensure technology translation and impact for biomedical research. To address this, previous work has focused on developing a stepwise differentiation protocol from hESC and iPSCs using defined additives and matrices15,23,24,25,26,27,28. By doing this, hepatocyte phenotype and reproducibility have been improved, permitting the semi-automation of the differentiation process19. The system presented is strengthened by its combination with&#160;off-the-shelf cell culture media and a facile hepatocyte differentiation system.
Previously, pluripotent cell density prior to the start of the differentiation protocol was highlighted as a key variable to achieve a homogenous population of hepatic progenitor cells26. Using this more refined procedure, it is possible to generate large numbers of stem cell-derived hepatic progenitors in a stepwise manner using a range of starting cell densities (Table 1). At day 5, definitive endoderm induction was validated by Sox17 staining (Figure 3). Efficient and robust differentiation into definitive endoderm was achieved with both tested ESC and iPSC lines, with more than 80% expressing Sox17 (Figure 3). At day 10, hepatic progenitors displayed a uniform cobblestone-like morphology, and liver stem cell markers were highly enriched for both AFP and HNF4&#945; (&#62;86%, Figure 4). Using a combination of manual and semi-automated technologies it was possible to perform differentiation in multiple plate formats19.
In its current form, cell differentiation is suitable for in vitro based experimentation. However, cell enrichment would likely be required before clinical application to ensure that a homogenous population of hepatic progenitors are prepared for delivery.
In conclusion, the protocol described here provides the field with a standardized approach to produce hepatic progenitors on a large scale. Future work will focus on the production of a new medium for subsequent HLC differentiation, maturation, and maintenance.

Liver disease represents a global health challenge, causing approximately 2 million deaths per year worldwide1. Although a number of model systems exist to study hepatic diseases and intervene clinically, the routine use of cell-based systems is limited by significant drawbacks (for a review see Szkolnicka et al.2). Advanced human pluripotent stem cell (hPSC) culture and somatic cell differentiation methods represent promising technologies to develop tools for basic biomedical research and renewable sources of differentiated cells for the clinic3,4.
To date, multiple protocols for hepatocyte-like cell (HLC) differentiation have been developed5,6,7,8. These protocols attempt to recreate aspects of human liver development by using a combination of small molecules and growth factors9,10. Most protocols consist of a stepwise differentiation process, where hPSCs are primed to definitive endoderm, followed by hepatic progenitor specification11,12,13, and ending with HLC specification. HLCs produced by these protocols display a mixture of fetal and adult phenotypes. This includes the expression of alpha fetoprotein (AFP), such as hepatocyte markers such as HNF4&#945; and&#160;albumin (ALB), as well as drug metabolizing capacity14,15,16. Between laboratories, HLC differentiation can vary; therefore, the development of standardized protocols is necessary. This will enable researchers to effectively generate and apply stem cell-derived HLCs on a large scale for basic and clinical research.
A hepatic progenitor differentiation system was developed that can be applied to both human embryonic and induced pluripotent stem cell lines using easy-to-follow guidelines. This procedure yields homogenous populations of hepatic progenitors in varying cultureware formats, ranging from cell culture flasks to 96 well plates. Provided below is the protocol to produce stem cell-derived hepatic progenitors in 24 and 96 well formats.
Cell density used in the protocol presented below is specified for one well of a 24 and 96 well plate respectively (see Table 1). Optimization of the starting cell number is required for the different cell culture plate formats and cell lines. Suggested starting cell density for protocol optimization is 2 x 105 cells/cm2. For density optimization, several cell densities can be tested by adding &#177; 50,000 cells/cm2 at a time.

<table><tbody><tr><td>DPBS with Calcium and Magnesium</td><td>ThermoFisher</td><td>14040133</td><td></td></tr><tr><td>Gentle cell dissociation reagent</td><td>STEMCELL Technologies</td><td>7174</td><td></td></tr><tr><td>Hoechst 33342 Ready Flow Reagent</td><td>thermofisher</td><td>R37165</td><td></td></tr><tr><td>Human Recombinant Laminin 521</td><td>BioLamina</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 Serum Alpha Fetoprotein ELISA</td><td>Alpha Diagnostics</td><td>500</td><td></td></tr><tr><td>mTeSR1 medium</td><td>STEMCELL Technologies</td><td>5850</td><td></td></tr><tr><td>Operetta High-Content Imaging System</td><td>PerkinElmer</td><td>HH12000000</td><td></td></tr><tr><td>PBS, no calcium, no magnesium</td><td>ThermoFisher</td><td>14190250</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Rho-associated kinase (ROCK)inhibitor Y27632</td><td>Sigma-Aldrich</td><td>Y0503-1MG</td><td></td></tr><tr><td>STEMdiff Definitive Endoderm Supplement CJ</td><td>STEMCELL Technologies</td><td></td><td></td></tr><tr><td>STEMdiff Definitive Endoderm Supplement MR</td><td>STEMCELL Technologies</td><td></td><td></td></tr><tr><td>STEMdiff Endoderm Basal Medium</td><td>STEMCELL Technologies</td><td></td><td></td></tr><tr><td>STEMdiff Hepatic Progenitor Medium</td><td>STEMCELL Technologies</td><td></td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma-Aldrich</td><td>P9416</td><td></td></tr><tr><td>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Albumin</td><td>Sigma-Aldrich</td><td>A6684</td><td>1:200 (mouse)</td></tr><tr><td>Alpha-fetoprotein</td><td>Sigma-Aldrich</td><td>A8452</td><td>1:400 (mouse)</td></tr><tr><td>HNF-4&amp;alpha;</td><td>Santa Cruz</td><td>sc-8987</td><td>1:400 (rabbit)</td></tr><tr><td>IgG</td><td>DAKO</td><td></td><td>1:400</td></tr><tr><td>Sox17</td><td>R&amp;amp;D Systems, Inc.</td><td>AF1924</td><td>1:200 (Goat)</td></tr><tr><td>&lt;strong&gt;Software&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Columbus Image Data Storage and Analysis system</td><td>PerkinElmer</td><td></td><td></td></tr></tbody></table>

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.

Hepatic progenitor differentiation from both hESC (H9) and hiPSC (P106) lines was performed following the stepwise protocol described in Figure 2. Here, pluripotent stem cells were seeded as single cells into LN-521-coated plates prior to the start of the differentiation. Cell confluency is the key for a robust and reproducible differentiation. Once the right confluency was achieved (Figure 2), differentiation was initiated. At day 5, definitive endoderm specification was assessed via Sox17 expression. In both cell lines, Sox17 was highly expressed with 80% &#177; 0.5% and 87.8% &#177; 0.5% SEM of Sox17-positive cells for H9 and P106, respectively (Figure 3). At day 10, hepatic progenitors displayed a cobblestone-like morphology (Figure 2). In addition, hepatic progenitor specification was assessed for HNF4&#945;, AFP, ALB, and cytokeratin-19 (CK19) expression as well as AFP and ALB protein secretion10,15,22 (Figure 4). Both H9 and P106 hepatic progenitor cultures expressed fetal hepatic markers such as HNF4&#945; (91% &#177; 0.5% and 90% &#177; 0.2%), AFP (89.7% &#177; 1.8% and 86% &#177; 1.2%), and CK19 (78.5% &#177; 3.2% and 83.6 &#177; 1.8%) (Figure 4). AFP secretion was detected at day 10 in both cell lines (32.4 &#177; 1.6 and 47.8 &#177; 5.9 ng/mL/mg/24 h) (Figure 5). Albumin synthesis was observed at lower levels (30.7% &#177; 1.8% and 27.2% &#177; 1.1%) (Figure 4) and was not detected via ELISA (Figure 5).
The protocol allowed the standardized production of hepatic progenitors from 24 well to 96 well plates. A semi-automated pipeline was employed to produce 96 well plates of hepatic progenitors from H9 and P106 cell lines as previously described17. Cell number variability and hepatic progenitor differentiation efficiency was assessed via quantification of HNF4&#945; expression. Cell segmentation was performed for protein quantification via immunofluorescence using a high content imaging instrument (Figure 1). At day 10, hepatic progenitors showed no significant variability across rows with &#62;94% of HNF4&#945;-positive cells per well for H9 and 97% HNF4&#945;-positive cells for P106 (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61256/61256fig1v2.jpg" /> 
Figure 1: Cell segmentation pipeline overview. (A) Using the original image, (B) nuclear staining was used for nuclei segmentation. (C) A nuclear segmentation quality control step based on shape and size was performed to only quantify clearly segmented nuclei. (D) Following this, positive HNF4&#945;-stained nuclei were quantified. (E) Finally, an intensity-based threshold was employed to identify HNF4&#945;-expressing cells. In C and E, green nuclei represent selected cells and magenta nuclei indicate discarded cells. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61256/61256fig1v2large.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/61256/61256fig2v2.jpg" /> 
Figure 2: Hepatic progenitor differentiation from hPSCs. (A) Schematic representation of the hepatic progenitor differentiation protocol. (B) Representative images highlighting the morphological changes during the differentiation. At day 0 (D0), hPSCs presented a packed monolayer of cells. Following this, hPSCs were primed into definitive endoderm on day 5 (D5). This was followed by hepatic progenitor differentiation on day 10 (D10). Hepatic progenitors displayed a cobblestone-like cell morphology. Scale bar = 75 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61256/61256fig2v2large.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/61256/61256fig3v2.jpg" /> 
Figure 3: Characterization of definitive endoderm specification. At day 5, cells were stained for Sox17, a definitive endoderm marker. The percentage of Sox17-positive cells was 80 &#177; 0.5% for H9 and 87.8 &#177; 0.5% for P106. Percentage quantification was based on 10 separate wells with 6 fields of view per well. Data are shown as the average &#177; SEM. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61256/61256fig3v2large.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/61256/61256fig4v3.jpg" /> 
Figure 4: Hepatic progenitor characterization. At day 10, hepatic progenitors were stained for hepatic markers (A) HNF4&#945;, (B) AFP, and (C) ALB. For H9, the percentage of positive cells were 91% &#177; 0.4%, 89.7% &#177; 1.8%, and 30.7% &#177; 1.8% for HNF4&#945;, AFP, and ALB, respectively. For P106, the percentage of positive cells were 90% &#177; 0.2%, 86% +/- 1.2%, and 27.2% &#177; 1.1% for HNF4&#945;, AFP, and ALB, respectively. (D) Cholangiocyte lineage potential was assessed via CK19 expression; H9-derived hepatic progenitors expressed 78.5% &#177; 3.2% CK19-positive cells, whereas 83.6% &#177; 1.8% of CK19-positive cells were observed for P106 hepatic progenitors. Immunoglobulin G (IgG) staining was used as a staining control. Percentage quantification was based on 10 separate wells with 6 fields of view per well. Data are shown as the average &#177; SEM. Scale bar = 50 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61256/61256fig4v3large.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/61256/61256fig5.jpg" /> 
Figure 5: Hepatic progenitor protein secretion analysis. The secretion of alpha fetoprotein (AFP) and albumin (ALB) was analyzed in hepatic progenitor cultures at day 10 in H9 and P109. The data represent three biological replicates and the error bars represent the SD. Secreted proteins were quantified from 24 h culture medium as nanograms of secreted protein per mL per mg of protein, n = 3; ND = not detected. <a href="https://www.jove.com/files/ftp_upload/61256/61256fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61256/61256fig6v2.jpg" /> 
Figure 6: Assessment of well-to-well variability in 96 well plate. (A) Visualization of a 96 well plate view of H9-derived hepatic progenitors stained with HNF4&#945;. (B) Quantification of the HNF4&#945;-positive cells. Average of cell number per well in rows, from six fields of view per well quantified. The average cell number across the plate was 94.81% &#177; 0.22 SEM HNF4&#945;-positive cells per well. No statistically significant differences were observed between wells. (C) Visualization of a 96 well plate view of P106-derived hepatic progenitors stained with HNF4&#945;. (D) Quantification of HNF4&#945;-positive cells. The average cell number per wells in rows, from six fields of view per well and quantified. The average cell number across the plate was 97.7% &#177; 0.57 SEM HNF4&#945;-positive cells per well. No statistically significant differences were observed between rows. Well H12 was used as an Immunoglobulin G (IgG) staining control. Scale bar = 1 mm. One-way ANOVA with Tukey&#39;s post-hoc statistical tests were employed. <a href="https://www.jove.com/files/ftp_upload/61256/61256fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1">
	<tbody>
		<tr>
			<td>Plate format</td>
			<td>Surface area (cm2)</td>
			<td>Cells per cm2</td>
			<td>Total cells per well</td>
			<td>Dispensing volume (mL)</td>
			<td>Cell concentration (cells/ml)</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>1.9</td>
			<td>210526</td>
			<td>400000</td>
			<td>0.5</td>
			<td>800000</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>0.32</td>
			<td>187500</td>
			<td>60000</td>
			<td>0.05</td>
			<td>1200000</td>
		</tr>
	</tbody>
</table>
Table 1: Recommended cell density for the different plate formats for the hPSC cell lines used in this protocol.

Watch this Scientific Journal Video about Hepatic Progenitor Specification from Pluripotent Stem Cells using a Defined Differentiation System at JoVE.com

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

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 ...

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4.6K Views.  University of Edinburgh. 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.

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

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

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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 ...

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.

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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.

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Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

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Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Differentiation of Human Pluripotent Stem Cells Into Pancreatic Beta-Cell Precursors in a 2D Culture System

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to diabetes. hPSC-derived insulin-secreting cells can be generated for cell therapy and disease modeling,&#160;however,&#160;with limited efficiency and functional properties. hPSC-derived pancreatic progenitors that are precursors to beta cells and other endocrine cells, when co-express the two transcription factors PDX1 and NKX6.1, specify the progenitors to functional, insulin-secreting beta cells both in vitro and in vivo. hPSC-derived pancreatic progenitors are currently used for cell therapy in type 1 diabetes patients as part of clinical trials. However, current procedures do not generate a high proportion of NKX6.1 and pancreatic progenitors, leading to co-generation of non-functional endocrine cells and few glucose-responsive, insulin-secreting cells. This work thus developed an enhanced protocol for generating hPSC-derived pancreatic progenitors that maximize the co-expression of PDX1 and NKX6.1 in a 2D monolayer. The factors such as cell density, availability of fresh matrix, and dissociation of hPSC-derived endodermal cells are modulated that augmented PDX1 and NKX6.1 levels in the generated pancreatic progenitors and minimized commitment to alternate hepatic lineage. The study highlights that manipulating the cell&#39;s physical environment during in vitro differentiation can impact lineage specification and gene expression. Therefore, the current optimized protocol facilitates the scalable generation of PDX1 and NKX6.1 co-expressing progenitors for cell therapy and disease modeling.

The present protocol describes an enhanced method to increase the co-expression of PDX1 and NKX6.1 transcription factors in pancreatic progenitors derived from human pluripotent stem cells (hPSCs) in planar monolayers. This is achieved by replenishing the fresh matrix, manipulating cell density, and dissociating the endodermal cells.

Human pluripotent stem cells (hPSCs) are an excellent tool for studying early pancreatic development and investigating the genetic contributors to ...

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 ...