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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	<li>Asrani, S. K., Devarbhavi, H., Eaton, J., Kamath, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+liver+diseases+in+the+world.">Burden of liver diseases in the world.</a> Journal of Hepatology. 70 (1), 151-171 (2019).</li><li>Szkolnicka, D., 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=Concise+Review:+Advances+in+Generating+Hepatocytes+from+Pluripotent+Stem+Cells+for+Translational+Medicine.">Concise Review: Advances in Generating Hepatocytes from Pluripotent Stem Cells for Translational Medicine.</a> Stem Cells Dayton Ohio. 34 (6), 1421-1426 (2016).</li><li>Heslop, J. A., Duncan, S. 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 border="0" cellpadding="0" cellspacing="0" style="width:719px;" width="717">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">DPBS with Calcium and Magnesium</td>
			<td style="width:171px;">ThermoFisher</td>
			<td style="width:119px;">14040133</td>
			<td style="width:107px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Gentle cell dissociation reagent</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td>7174</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hoechst 33342 Ready Flow Reagent</td>
			<td style="width:171px;">thermofisher</td>
			<td>R37165</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Recombinant Laminin 521</td>
			<td>BioLamina</td>
			<td>LN521-02</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Serum Albumin ELISA</td>
			<td>Alpha Diagnostics</td>
			<td>1190</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Human Serum Alpha Fetoprotein ELISA</td>
			<td>Alpha Diagnostics</td>
			<td>500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">mTeSR1 medium</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td>5850</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Operetta High-Content Imaging System</td>
			<td>PerkinElmer</td>
			<td>HH12000000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">PBS, no calcium, no magnesium</td>
			<td style="width:171px;">ThermoFisher</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:171px;">Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rho-associated kinase (ROCK)inhibitor Y27632</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503-1MG</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">STEMdiff Definitive Endoderm Supplement CJ</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">STEMdiff Definitive Endoderm Supplement MR</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">STEMdiff Endoderm Basal Medium</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">STEMdiff Hepatic Progenitor Medium</td>
			<td style="width:171px;">STEMCELL Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:323px;">TWEEN 20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Antibodies</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A6684</td>
			<td style="width:107px;">1:200 (mouse)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Alpha-fetoprotein</td>
			<td>Sigma-Aldrich</td>
			<td>A8452</td>
			<td style="width:107px;">1:400 (mouse)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HNF-4&#945;</td>
			<td style="width:171px;">Santa Cruz</td>
			<td>sc-8987</td>
			<td>1:400 (rabbit)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">IgG</td>
			<td style="width:171px;">DAKO</td>
			<td style="width:119px;"></td>
			<td>1:400</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sox17</td>
			<td>R&#38;D Systems, Inc.</td>
			<td>AF1924</td>
			<td>1:200 (Goat)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:323px;">Software</td>
			<td style="width:171px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:323px;">Columbus Image Data Storage and Analysis system</td>
			<td>PerkinElmer</td>
			<td></td>
			<td style="width:107px;"></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 ...

hepatic-progenitor-specification-from-pluripotent-stem-cells-using

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JoVE Journal

Biology

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

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology&#8212;provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications&#8212;for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.

We describe a protocol for deriving lentiviral-based reprogrammed and characterized factor-free human induced pluripotent stem cells and conversion into putative clinical-grade conditions.

Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic ...

Evaluating the Angiogenetic Properties of Ovarian Cancer Stem-Like Cells using the Three-Dimensional Co-Culture System, NICO-1

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular matrix. The ability of CSCs to participate in the development of endothelium constitutes an important characteristic that directly contributes to the general understanding of the mechanisms of tumorigenesis and tumor metastasis. The purpose of this work is to establish a reproducible methodology to investigate the tumor-initiation capability of ovarian cancer stem cells (OCSCs). Herein, we examined the neovascularization mechanism between endothelial cells and OCSCs along with the morphological changes of endothelial cells using the in vitro co-culture model NICO-1. This protocol allows visualization of the neovascularization step surrounding the OCSCs in a time course manner. The technique can provide insight regarding the angiogenetic properties of OCSCs in tumor metastasis.

Ovarian cancer stem cells (OCSC) are responsible for cancer initiation, recurrence, therapeutic resistance, and metastasis. The OCSC vascular niche is considered to promote self-renewal of OCSCs, leading to chemoresistance. This protocol provides the basis for establishing a reproducible OCSC vascular niche model in vitro.

Cancer stem cells (CSCs) reside in a supportive niche, constituting a microenvironment comprised of adjacent stromal cells, vessels, and extracellular ...

Characterizing Surface Antigens and Proliferation Ability in hiPSC-Driving MSCs

Comparison of Two Representative Methods for Differentiation of Human Induced Pluripotent Stem Cells into Mesenchymal Stromal Cells

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are restricted by limited donation, quality variations, and biosafety, the past 10 years have seen a great rise in efforts to generate MSCs from human induced pluripotent stem cells (hiPSCs). Past and recent efforts in the differentiation of hiPSCs into MSCs have been centered around two culture methodologies: (1) the formation of embryoid bodies (EBs) and (2) the use of monolayer culture. This protocol describes these two representative methods in deriving MSC from hiPSCs. Each method presents its advantages and disadvantages, including time, cost, cell proliferation ability, the expression of MSC markers, and their capability of differentiation in vitro. This protocol demonstrates that both methods can derive mature and functional MSCs from hiPSCs. The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation, while the EB method is characterized by lower time consumption.

Author Spotlight: Advancements in iPSCs and Genetic Disease Research 

This protocol describes and compares two representative methods for differentiating hiPSCs into mesenchymal stromal cells (MSCs). The monolayer method is characterized by lower cost, simpler operation, and easier osteogenic differentiation. The embryoid bodies (EBs) method is characterized by lower time consumption.

Mesenchymal stromal cells (MSCs) are adult pluripotent stem cells which have been widely used in regenerative medicine. As somatic tissue-derived MSCs are ...

Purification of Human iPSC-Derived Hepatocytes by Mitochondrial-ALCAM Sorting

Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely diseased organs due to their unlimited proliferation and pluripotent properties. However, differentiating human ES/iPS cells into 100% pure target cell types is challenging due to their high sensitivity to the environment. Tumorigenesis after transplantation is caused by contaminated, proliferating, and undifferentiated cells, making high-purification technology essential for the safe realization of regenerative medicine. To mitigate the risk of tumorigenesis, a high-purification technology has been developed for human iPS cell-derived hepatocytes. The method employs FACS (fluorescence-activated cell sorting) using a combination of high mitochondrial content and the cell-surface marker ALCAM (activated leukocyte cell adhesion molecule) without genetic modification. 97% &plusmn; 0.38% (n = 5) of the purified hepatocytes using this method exhibited albumin protein expression. This article aims to provide detailed procedures for this method, as applied to the most current two-dimensional differentiation method for human iPS cells into hepatocytes.

Author Spotlight: Advancing Hepatocyte Purification from Human Induced Pluripotent Stem Cells for Regenerative Medicine

Hepatocytes derived from pluripotent stem cells can be purified through cell sorting, using a combination of mitochondrial and activated leukocyte cell adhesion molecule (ALCAM, also known as CD166) staining.

Human embryonic stem (ES) and induced pluripotent stem (iPS) cells have potential applications in cell-based regenerative medicine for treating severely ...

Differentiating Human Pluripotent Stem Cells into Pancreatic β-Cells

Optimized Protocol for Generating Functional Pancreatic Insulin-secreting Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) can differentiate into any kind of cell, making them an excellent alternative source of human pancreatic &#946;-cells. hPSCs can either be embryonic stem cells (hESCs) derived from the blastocyst or induced pluripotent cells (hiPSCs) generated directly from somatic cells using a reprogramming process. Here a video-based protocol is presented to outline the optimal culture and passage conditions for hPSCs, prior to their differentiation and subsequent generation of insulin-producing pancreatic cells. This methodology follows the six-stage process for &#946;-cell directed differentiation, wherein hPSCs differentiate into definitive endoderm (DE), primitive gut tube, posterior foregut fate, pancreatic progenitors, pancreatic endocrine progenitors, and ultimately pancreatic &#946;-cells. It is noteworthy that this differentiation methodology takes a period of 27 days to generate human pancreatic &#946;-cells. The potential of insulin secretion was evaluated through two experiments, which included immunostaining and glucose-stimulated insulin secretion.

Author Spotlight: Advancements and Challenges in &#946;-Cells Differentiation from Pluripotent Stem Cells

This article presents a protocol for directed differentiation and functional analysis of &#946;-cell like cells. We describe optimal culture conditions and passages for human pluripotent stem cells before generating insulin-producing pancreatic cells. The six-stage differentiation progresses from definitive endoderm formation to functional &#946;-cell like cells secreting insulin in response to glucose.