This work was supported with awards from the UK Regenerative Medicine Platform (MRC MR/L022974/1 and MR/K026666/1) and a China Scholarship.

NOTE: Vendor information for all reagents used in this protocol has been listed in Table 1. All media/plates should be sterile and at least at room temperature when cells are to have direct contact with them.
1. Passaging Human Pluripotent Stem Cells (hPSCs) onto Laminin 521
NOTE: The cell passaging procedure described below is based on single cells and is ideal for the derivation of a homogenous population of hepatocyte-like cells from hPSCs. Colony plating is also applicable and has been described previously24.
<ol>
	<li>Prepare laminin-coated plates as needed.
	<ol>
		<li>Thaw the 100 &#181;g/mL stock of recombinant laminin 521 (LN-521) at 4 &#176;C.</li>
		<li>Dilute the thawed LN-521 in ice-cold 1x DPBS (with Ca2+/Mg2+) to make a 5 &#181;g/mL solution.</li>
		<li>Add 1 mL of 5 &#181;g/mL LN-521 solution to coat one well of a 6-well plate and rock to spread it evenly in the well.</li>
		<li>Incubate the plates in a 37 &#176;C/5% CO2 cell culture incubator for 2 - 4 h for urgent use or in a 4 &#176;C refrigerator overnight.</li>
		<li>Store the laminin-coated plates in a 4 &#176;C refrigerator as required. Keep the plates on a flat surface and seal them to avoid evaporation and contamination. 
		NOTE: Never let the coated wells dry out; top them up with extra 1x DPBS (with Ca2+/Mg2+) if required. Use the plates within 2 weeks.</li>
	</ol>
	</li>
	<li>Allow the required number of pre-coated plates to reach room temperature before use or incubate the plates at 37 &#176;C for 0.5 - 1 h.</li>
	<li>Carefully aspirate the coating LN-521 solution without damaging the coated surface. Note: It is critical not to damage the coated surface before seeding cells on it.</li>
	<li>Immediately add 1 mL of pre-warmed-mTeSR1 medium supplemented with 10 &#181;M Rho-associated kinase (ROCK) inhibitor Y27632 to one well of a 6-well plate. Leave the plate in the cell culture incubator to receive cells. 
	NOTE: Never allow the laminin-coated wells to dry.</li>
	<li>Aspirate the medium from well-maintained hPSCs at about 75% to 85% confluency. Wash the cells from one well of a 6-well plate once with 1 mL of room-temperature 1x DPBS (no Ca2+/Mg2+).</li>
	<li>Add 0.5 mL of 1x Accutase to the cells and incubate at 37 &#176;C for 6 - 8 min to dissociate the cells. 
	NOTE: To check whether the digestion is long enough or not, gently tap the plate and check whether the cells can detach easily. If yes, then it is time to stop the enzymatic reaction; if not, extend the digestion for an extra 1 - 2 min.</li>
	<li>Terminate the dissociation by adding 2 mL of fresh mTeSR1 medium supplemented with 10 &#181;M Y27632 to the cells. Pipette up and down using a P1000 tip several times to make a single-cell suspension.</li>
	<li>Count the viable cells using a hemocytometer. Use Trypan Blue to stain and exclude dead cells14</li>
	<li>Calculate the total number of cells needed. For routine hPSC passaging, seed 4 x 105 to 5 x 105 cells per well of a 6-well plate (i.e., 4.21 x 104 to 5.26 x 104 per cm2). For passaging hPSCs for hepatocyte differentiation, seed 6.5 x 105 to 7.5 x 105 (i.e., 6.84 x 104 to 7.89 x 104 per cm2) cells per well of a 6-well plate. 
	NOTE: The seeding density for each cell line might need minor optimization based on the empirical density given here for hepatic differentiation.</li>
	<li>Transfer the needed cell suspension into a sterile 15-mL or 50-mL centrifuge tube and centrifuge at 115 x g for 3 min at room temperature.</li>
	<li>Aspirate the supernatant slowly and then resuspend the cell pellet in fresh, warm mTeSR1 medium supplemented with 10 &#181;M Rho-associated kinase (ROCK) inhibitor Y27632, using adequate medium to make the desired cell density. 
	NOTE: The use of ROCK inhibitor is highly recommended in order to enhance cell attachment and survival rate.</li>
	<li>Seed the cells to the prepared plates and rock them back and forth and side to side to evenly distribute the cells. 
	NOTE: It is critical to ensure that cells are distributed evenly in the wells whether the plate is for routine cell culture or hepatocyte differentiation experimentation.</li>
	<li>Place the plates in the cell incubator and maintain the cells at 37 &#176;C/5% CO2 for 24 h to allow them to attach and recover.</li>
	<li>Examine the cells the next day and withdraw ROCK inhibitor if cell-cell contact has been established. Maintain the cells in mTeSR1 medium for routine culture or switch to differentiation medium as needed. 
	NOTE: If the cells were seeded with the mentioned density, the confluency should be ideal for routine maintenance or hepatocyte differentiation.</li>
</ol>
2. Differentiating hPSCs to Hepatocyte-like Cells on Recombinant Laminins
<ol>
	<li>Prepare differentiation medium.
	<ol>
		<li>Make human Activin A stock solution: dissolve human Activin A powder to make a 100 &#181;g/mL stock solution in sterile 0.2% bovine serum albumin (BSA)/DPBS. Make small aliquots and store them at -20 &#176;C. Use at 1:1,000.</li>
		<li>Make mouse Wnt 3a stock solution: dissolve mouse Wnt 3a powder to make a 10 &#181;g/mL stock solution in sterile 0.2% BSA/DPBS. Make small aliquots and store them at -20 &#176;C. Use at 1:200.</li>
		<li>Make human hepatocyte growth factor (HGF) stock solution: dissolve human HGF powder to make a 10 &#181;g/mL stock solution in sterile 0.2% BSA/DPBS. Make small aliquots and store them at -20 &#176;C. Use at 1:1,000.</li>
		<li>Make Oncostatin M (OSM) stock solution: dissolve Oncostatin M (OSM) powder to make a 20 &#181;g/mL stock solution in sterile 0.2% BSA/DPBS. Make small aliquots and store them at -20 &#176;C. Use at 1:1,000.</li>
		<li>Make 500 mL of endoderm-priming stock medium: 2% B27 supplement (50x, minus vitamin A) and 1% penicillin/streptomycin (final concentrations at 100 IU/mL and 100 &#181;g/mL, respectively); top up to 500 mL using Roswell Park Memorial Institute 1640 (RPMI 1640) basal medium. NOTE: Store the stock at 4 &#176;C and use within two weeks. Aliquot medium from the stock and add fresh Activin A and Wnt 3a (final concentrations at 100 ng/mL and 50 ng/mL, respectively) at each medium change.</li>
		<li>Make 500 mL of KSR/DMSO differentiation medium: 80% knockout DMEM (KO-DMEM), 20% knockout serum replacement (KSR), 0.5% GlutaMAX, 1% non-essential amino acids (NEAA), 0.1 mM beta-mercaptoethanol, 1% DMSO, and 1% penicillin/streptomycin (final concentrations at 100 IU/mL and 100 &#181;g/mL, respectively). Filter under vacuum. Store at 4 &#176;C and use within two weeks.</li>
		<li>Make 500 mL of HepatoZYME maturation medium: 1% GlutaMAX, 10 &#181;M hydrocortisone 21-hemisuccinate sodium salt (HCC), and 1% penicillin/streptomycin (final concentrations at 100 IU/mL and 100 &#181;g/mL, respectively); top up to 500 mL using HepatoZYME basal medium. NOTE: Store the stock at 4 &#176;C and use within two weeks. Aliquot medium from the stock and add fresh HGF and OSM (final concentrations at 10 ng/mL and 20 ng/mL, respectively) for each medium change.</li>
	</ol>
	</li>
	<li>Seed hPSCs for hepatocyte differentiation on LN-521, as described in section 1. If LN-521/LN-111 is to be used as the substrate, coat the plates with LN-521 and LN-111 (1:3 ratio) at the final laminin concentration of 5 &#181;g/mL; the rest treatment should be the same as pure LN-521-coated plates. 
	NOTE: LN-521/LN-111 is not ideal for routine culture of hPSCs; it is only used for differentiation experiments.</li>
	<li>Check the cell confluency 24 h after seeding. Initiate cellular differentiation once the cell confluency reaches about 40%. Remove the spent mTeSR1 medium and add fresh endoderm-priming medium supplemented with 100 ng/mL Activin A and 50 ng/mL Wnt 3a. Call this differentiation day 1. 
	NOTE: It is highly recommended to initiate the differentiation the day after seeding the cells.</li>
	<li>Change the endoderm-priming medium every 24 h for 3 days for human embryonic stem cells (hESCs). As for human induced pluripotent stem cells (hiPSCs), extend this stage for 2 more days to prime the cells to definitive endoderm-like cells, but use the endoderm-priming medium supplemented only with 100 ng/mL Activin A for these two days12. 
	NOTE: To ensure successful endoderm specification, one can examine the expression of endodermal markers, such as FOXA2 and SOX17. According to immunofluorescence staining, over 80% of the derived cells are positive for both markers in our lab.</li>
	<li>Switch to KSR/DMSO differentiation medium on day 4 (for hESCs)/day 6 (for hiPSCs). Change the medium daily for the first 3 days and then on the fifth day of this differentiation stage. 
	NOTE: No feeding is needed on the fourth day of this differentiation stage. Use unsupplemented KO-DMEM to wash the cells once before medium change if there are many dead cells. To check whether the differentiation for this stage is successful or not, one can test the expression of hepatic progenitor cell markers, such as AFP, CK19, and HNF4A. Nearly 90% of the cells will be positive for these markers based on our experience.</li>
	<li>After 5 days of the KSR/DMSO differentiation stage, switch to the HepatoZYME maturation stage. Wash the cells once with plain HepatoZYME basal medium after removing the KSR/DMSO medium. Add HepatoZYME maturation medium supplemented with 10 ng/mL HGF and 20 ng/mL OSM.</li>
	<li>Change the medium every 48 h for 7 - 10 days, at which point the cells are ready for standard characterization or further use. 
	NOTE: Hepatocyte marker expression examinations, metabolic function tests (such as cytochrome P450 activity), urea and albumin secretion tests, glycogen storage tests, and Indocyanine green (ICG) uptake tests are typical characterization methods. 
	NOTE: The timeline of the differentiation protocol is shown in Figure 5. In our lab, we routinely check the derived HLCs&#39; hepatocyte-specific marker expression levels, albumin secretion, and cytochrome P450 (CYP) 3A and 1A2 activity.</li>
</ol>

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Dr. David C. Hay is a co-founder and director of Stemnovate Limited.

To advance human pluripotent stem cell research and translational medicine, xeno-free systems that comply with current good manufacturing practice guidelines are required. Key to any differentiation process is the extracellular matrix (ECM). The ECM not only supports cell attachment, but also provides access to key signaling factors, influencing cell determination and phenotype25,26.
Laminins are multifunctional extracellular matrix proteins in vivo. In the liver, laminin secretion is crucial for liver regeneration after a partial hepatectomy27 and is required for hepatic progenitor cell maintenance28. The importance of laminins in liver maintenance and regeneration was the basis for testing commercially available recombinant human laminins in our hepatocyte differentiation system.
Superior hepatocyte differentiation was achieved on LN-521 and LN-521/LN-111 substrates when compared to Matrigel. Derived HLCs were clearly polarized and organized in the dish, and their cellular function was significantly improved when compared to their gelatinous protein mixture counterparts. Underlying these improvements was the downregulation of contaminating colon-, fibroblast- and stem cell-associated genes on the laminins, as well as a decrease in cell proliferation and migration-associated gene expression10.
In conclusion, the protocol described here generates hepatocyte-like cells that are closer in nature to adult human hepatocytes. The process is reproducible, amenable to automation, and can be cost-effectively scaled for application. Importantly, batch-to-batch variation has been significantly decreased in comparison to techniques that use Matrigel, resulting in an improved differentiation system for researchers within this field.

Primary human tissue and the derivative cell types are regularly used, both for cell-based screening and in the clinic. However, access to these cells is severely limited due to insufficient organ donation and loss of cell phenotypes post-isolation1. hPSCs represent a promising alternative to primary tissue and facilitate the generation of genetically defined and renewable human somatic cells. Hepatocyte-like cells (HLCs) derived from hPSCs have already shown promise in this field. HLCs resemble primary human hepatocytes in various aspects, including cell morphology, hepatocyte gene expression, metabolic function, and sensitivity to drugs and viruses2,3,4,5,6,7,8. In addition, the unlimited proliferation and self-renewal capacity of both research- and GMP-grade hPSCs facilitates their application9,10.
Over a decade of research has produced a number of efficient hepatocyte differentiation procedures2,3,5,11,12,13,14,15,16,17,18,19,20. However, most of these systems use undefined components and/or viral transduction to drive hepatocellular specification. To improve the reliability of the technology at scale, it is important to develop a robust hepatocyte differentiation system that is truly defined, xeno-free, and GMP-compatible.
Laminins (LNs) are important extracellular matrix proteins that can influence cell adhesion, proliferation, migration, and differentiation. Laminins are heterotrimeric glycoproteins composed of one &#945;, one &#946;, and one &#947; chain. Recently, recombinant human laminins have been produced and used in cell biology. LN-511 has been shown to support the maintenance of hPSCs21, while a mixture of LN-521 and E-cadherin allows clonal derivation and the expansion of human embryonic stem cells22. LN-111, on the other hand, supports the maintenance of hepatoblast-like cells derived from hPSCs23. However, before our report, laminins 521 and 111 had not been utilized to generate HLCs with mature characteristics from hPSCs10.
Here, we detail procedures for culturing hPSCs on LN-521 and differentiating them on either LN-521 or a blend of LN-521 and LN-111 (LN-521/LN-111). We optimized the differentiation protocol using single-cell seeding to generate a highly reproducible and homogenous monolayer of HLCs in many formats14. We believe that our defined differentiation system represents a simple and cost-effective method to manufacture active HLCs for application, representing a significant step forward in the field.

<table><tbody><tr><td>Human Recombinant Laminin 521</td><td>BioLamina</td><td>LN521-02</td><td></td></tr><tr><td>Human Recombinant Laminin 111</td><td>BioLamina</td><td>LN111-02</td><td></td></tr><tr><td>Recombinant mouse Wnt3a</td><td>R&amp;amp;D Systems</td><td>1324-WN-500/CF</td><td></td></tr><tr><td>Human Activin A</td><td>Peprotech</td><td>120-14E</td><td></td></tr><tr><td>Human Hepatocyte Growth Factor</td><td>Peprotech</td><td>100-39</td><td></td></tr><tr><td>Human Oncostatin M</td><td>Peprotech</td><td>300-10</td><td></td></tr><tr><td>Rho-associated kinase (ROCK) inhibitor Y27632&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Y0503-1MG</td><td></td></tr><tr><td>Hydrocortisone 21-hemisuccinate sodium salt</td><td>Sigma-Aldrich</td><td>H4881</td><td></td></tr><tr><td>DMSO</td><td>Sigma-Aldrich</td><td>D5879</td><td></td></tr><tr><td>mTeSR1 medium</td><td>STEMCELL Technologies</td><td>05850</td><td></td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>21875</td><td></td></tr><tr><td>Knockout DMEM</td><td>Life Technologies</td><td>10829</td><td></td></tr><tr><td>HepatoZYME</td><td>Life Technologies</td><td>17705</td><td></td></tr><tr><td>B27 supplement</td><td>Life Technologies</td><td>12587-010</td><td></td></tr><tr><td>Knockout Serum Replacement</td><td>Life Technologies</td><td>10828</td><td></td></tr><tr><td>GlutaMax</td><td>Life Technologies</td><td>35050</td><td></td></tr><tr><td>Non-essential amino acids</td><td>Life Technologies</td><td>11140</td><td></td></tr><tr><td>2-mercaptoethanol</td><td>Life Technologies</td><td>31350</td><td></td></tr><tr><td>Accutase</td><td>Millipore</td><td>SCR005</td><td></td></tr><tr><td>DPBS with Calcium and Magnesium</td><td>ThermoFisher</td><td>14040133</td><td></td></tr></tbody></table>

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.

Hepatocellular Differentiation from hPSCs
One human embryonic stem cell line, H9, and one human induced pluripotent stem cell line, 33D6, were used for hepatocyte differentiation. The results in Figures 1-3 are from H9 cells, while those in Figure 4 are from 33D6 cells. Single cells seeded onto laminins established cell-cell contact after 24 h. After the cells reached around 40% confluency, the differentiation process was initiated (Figure 1A and Figure 4A). On laminins (both LN-521 and LN-521/LN-111), these cells went through sequential morphological changes and gave rise to polarized HLCs (Figure 1 and Figure 4A).
Hepatocyte-like Cell Characterization
Day 18 HLCs were collected and assessed for the expression of representative hepatocyte markers, HNF4A and ALB (Figure 2A). Immunostaining of day 18 HLCs showed that nearly 90% of the cells expressed HNF4&#945; (Figure 2B). These cells polarized on laminins and exhibited a polygonal appearance, as marked by E-cadherin and F-actin expression (Figure 2B).
Cytochrome P450 (CYP) activity was also assessed. The CYP450s conduct an important metabolic function of hepatocytes. Day 18 HLCs derived on a gelatinous protein mixture, such as Matrigel, LN-521, or LN-521/LN-111, were tested for CYP3A activity. HLCs demonstrated significantly higher CYP3A activity on laminin substrates than on matrigel (Figure 3). Importantly, when compared to commercial human primary hepatocytes (HU1339) re-plated on these substrates, HLCs have nearly 10 times higher levels of CYP3A activity10.
The differentiation of hiPSCs was similar to that of hESCs. The cells exhibited sequential changes in appearance (Figure 4A). Derived HLCs expressed a key hepatocyte transcription factor, HNF4&#945; (Figure 4B), and possessed CYP3A activity and secreted albumin (Figure 4C and D). Notably, HLCs derived from 33D6 displayed reduced CYP3A in comparison to H9 cells derived HLCs (Figure 3), but it was still comparable to human primary hepatocytes10. However, the albumin secretion of these HLCs was much lower than in primary hepatocytes10.
<img alt="Figure 1" src="/files/ftp_upload/55355/55355fig1.jpg" /> 
Figure 1: The Sequential Morphological Changes during Hepatic Differentiation. (A) Undifferentiated hESCs seeded as single cells reached around 40% confluency 24 h after seeding. (B) After priming, cells exhibited the typical endodermal morphology on day 4. (C) Upon reaching the hepatoblast-like stage, they showed a clear polygonal shape on day 9. (D) After the maturation stage, polarized HLCs were ready for further characterization shown here at day 18. Scale bar = 200 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55355/55355fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55355/55355fig2.jpg" /> 
Figure 2: Characterization of HLCs. (A) Gene expression of hepatocyte-specific markers, HNF4A (left) and ALB (right). The expression level was analyzed using day 18 HLCs derived from hESCs on both LN-521 and LN-521/LN-111, and it was normalized to the housekeeping gene GAPDH and expressed relative to hESCs. The results represent three biological replicates, and the error bars represent standard deviation (SD). * p &#60; 0.05, ** p &#60; 0.01; unpaired t-test. (B) Protein expression of a key hepatic marker, HNF4&#945;, and polarization markers, E-cadherin and F-actin. Day 18 HLCs on LN-521 and LN-521/LN-111 were stained for the above markers and counterstained with Hoechst 33342. A negative control was performed with the corresponding immunoglobulin G (IgG). The percentage of HNF4&#945;-positive cells and the SD is shown. This was calculated from four random fields of view. Images were taken at 20X magnification. Scale bar = 100 &#181;m. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55355/55355fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55355/55355fig3.jpg" /> 
Figure 3: Metabolic Function Characterization of HLCs. Cytochrome P450 activity of CYP3A of cells cultured on Matrigel (MG), LN-521, or LN-521/LN-111 was tested. The data represent three biological replicates, and the error bars represent SD. ** p &#60; 0.01; one-way ANOVA with Tukey&#39;s post-hoc test. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55355/55355fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55355/55355fig4.jpg" /> 
Figure 4: Standard Characterization of HLCs. hiPSCs cultured on LN-521 were differentiated into HLCs. Standard characterization tests were performed on day 17 HLCs. (A) The sequential morphology of the cells during hepatic differentiation; the time points shown represent cells on days 1, 4, 9, and 17. (B) Immunostaining of HNF4&#945; expression. The percentage of positive cells and the SD is shown based on four random fields of view. Images were taken at 20X magnification. Scale bar = 100 &#181;m. (C) CYP3A activity on day 17 HLCs. The data represent six biological replicates, and the error bar represents the SD. (D) Albumin secretion of derived HLCs over 24 h in culture. The data represent four biological replicates, and the error bar represents SD. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55355/55355fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55355/55355fig5.jpg" /> 
Figure 5: Schematic Timeline of the Differentiation Protocol. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55355/55355fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a> 
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Watch this Scientific Journal Video about Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells at JoVE.com

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

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

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

defined-scalable-generation-hepatocyte-like-cells-from-human

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

12.4K Views.  University of Edinburgh. 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.

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

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

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

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

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

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

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

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

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

Efficient Differentiation of Human Pluripotent Stem Cells into Liver Cells

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

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

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

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

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

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

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

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

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

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

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

Biology

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

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

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

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

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.

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Bioengineering

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

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

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

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

Immunology and Infection

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

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

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

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Monitoring Pluripotent Stem Cell Differentiation by Image-Based ML Strategy

A Live-cell Image-Based Machine Learning Strategy to Monitor Pluripotent Stem Cell Differentiation

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available PSC-to-functional cell differentiation systems are impeded by problems of severe line-to-line and batch-to-batch variability. Precise control of cell differentiation in real time is therefore important. In this protocol, we describe a non-invasive and intelligent strategy that overcomes the variability in cell differentiation by using bright-field image-based machine learning. Taking PSC-to-cardiomyocyte differentiation as an example, this methodology provides detailed information for control of the initial PSC state, early assessment and intervention in differentiation conditions, and elimination of the misdifferentiated cell contamination, together realizing consistently high-quality differentiation from PSCs to functional cells. In principle, this strategy can be extended to other cell differentiation or reprogramming systems with multiple steps to support cell manufacturing, as well as to further our understanding of the mechanisms during cell fate conversion.

Author Spotlight: Enhancing PSC-to-Functional Cell Differentiation Using ML Models Based on Live-Cell Bright-Field Imaging

Available pluripotent stem cell (PSC)-to-functional cell differentiation systems are currently impeded by problems of severe line-to-line and batch-to-batch variability. Here, using cardiac differentiation as the main example, we present a protocol to intelligently monitor and modulate the process of PSC differentiation based on image-based machine learning.

Pluripotent stem cell (PSC) technologies have been widely used in drug discovery, disease modeling, and regenerative medicine.&#160;However, available ...