This work was supported by the Ministry of Education, Culture, Sports, Science and Technology grant number [23390072].

This study used commercially obtained human iPS cells (253G1 strain) (see Table of Materials).
1. Maintenance of human iPS cells
<ol>
	<li>Maintain human iPS cells in feeder-free conditions in AK02 medium on culture dishes coated with 0.25 &#181;g/cm2 iMatrix-511 (see Table of Materials).</li>
</ol>
2. Hepatic differentiation of human iPS cells in 2D cultures
NOTE: Refer to Figure 1A for the daily schedule of hepatic differentiation. Throughout the process, use 6-well culture plates and add 2 mL of the designated wash or culture medium to each well. Maintain the cell culture condition at 100% humidity, 5% CO2, and 20% O2 at 37 &#176;C.
<ol>
	<li>Coat each well of a 6-well plate with 1 mL of basement membrane matrix (diluted 1/25 in PBS (-)), and incubate at 37 &#176;C for 1 h.</li>
	<li>Passage human iPS cells onto the coated dish at a density of 20,000-30,000 cells/cm&#178; using AK02N medium supplemented with 10 &#181;M ROCK inhibitor (Y-27632) (see Table of Materials). Replace the medium without Y-27632 the following day and culture for an additional 2 days.</li>
	<li>Wash the cells once with RPMI-1640 medium. Initiate endoderm differentiation by replacing the medium with RPMI-B27-Glu (RPMI-1640 + 2% B27 Insulin + 1% Dipeptide L-Alanyl-L-Glutamine) supplemented with 3-6 &#181;M CHIR-99021 and 100 ng/mL Activin A for 24 h (see Table of Materials).</li>
	<li>Replace the medium with RPMI-B27-Glu supplemented with 50 ng/mL Activin A for 24 h.</li>
	<li>To induce hepatic progenitor differentiation, replace the medium with RPMI-B27-Glu supplemented with 1% dimethyl sulfoxide (DMSO) for 5 days.</li>
	<li>To promote hepatocyte maturation, switch to hepatocyte maturation medium: Leibovitz&#39;s L-15 medium containing 10 &#181;M hydrocortisone 21-hemisuccinate, 8.3% tryptose phosphate broth, 100 nM dexamethasone (DEX), 50 &#181;g/mL sodium-L-ascorbate, 0.58% insulin-transferrin-selenium (ITS), 8.3% fetal bovine serum (FBS), 2 mM Dipeptide L-Alanyl-L-Glutamine, and 100 nM N-Hexanoic-Try-Ile-(6)-amino Hexanoic amide (Dihexa) (see Table of Materials).</li>
	<li>Change the medium every 2 days for 20 days. Sort the cells approximately 27 days after initiating differentiation.</li>
</ol>
3. Cell preparation for purification of human iPS cells-derived hepatocytes
<ol>
	<li>Wash hepatic differentiated cells with 2 mL PBS (-) per well.</li>
	<li>Add 1 mL enzyme solution per well, which includes 0.1% collagenase, 0.083% trypsin, 10 &#181;M ROCK inhibitor (Y-27632), 20 nM ciclosporin A, and 50 &#181;g/mL sodium-L-ascorbate in Ads buffer (116 mM NaCl, 12.5 mM NaH2PO4, 20 mM HEPES, 5.4 mM KCl, 5.6 mM glucose, and 0.8 mM MgSO4; pH 7.35) (see Table of Materials). Detach cells from the plates by horizontally rotating at 37 &#176;C for about 2 h.</li>
	<li>After confirming that cells are detached and rounded under a microscope, disperse them into single cells by gentle pipetting, and collect them in a 50 mL tube.</li>
	<li>Centrifuge cells at 200 x g for 5 min at 18 &#176;C to pellet them. Remove the supernatant using an aspirator.</li>
	<li>Stain the mitochondria of the cells by dispersing them in 5 mL of hepatocyte maturation medium containing 100 nM TMRM (tetramethylrhodamine, methl ester) (see Table of Materials). Incubate for 30 min at 37 &#176;C while protecting from light by wrapping in aluminum foil.</li>
	<li>Centrifuge the cells at 200 x g for 5 min at 18 &#176;C to pellet them. Remove the supernatant.</li>
	<li>Resuspend the cells with 1-5 mL of cold Ads buffer containing 2% FBS.</li>
	<li>Count the cell number using a 0.4% Trypan blue solution and a cell count plate.</li>
	<li>Centrifuge the cells at 200 x g for 5 min at 4 &#176;C to pellet them. Remove the supernatant.</li>
	<li>Dilute an anti-ALCAM antibody (primary antibody, see Table of Materials) at a 1:50 ratio in Ads buffer, and add 100 &#181;L of the diluted antibody per 106 cells. Stain the cells for 50 min on ice. Prepare cells that are not stained with the primary antibody as a negative control.</li>
	<li>Wash the cells with cold Ads buffer containing 2% FBS twice.</li>
	<li>Dilute an Alexa Fluor 488 donkey anti-goat IgG (secondary antibody, see Table of Materials) at a 1:100 ratio in Ads buffer, and add 100 &#181;L of the diluted antibody per 106 cells. Stain the cells for 30 min on ice. Stain cells that were not stained with the primary antibody using the secondary antibody.</li>
	<li>Wash the cells with cold Ads buffer containing 2% FBS twice.</li>
	<li>Resuspend the cells in Ads buffer and filter them through a snap cap cell strainer (35 &#181;m mesh) just before sorting by FACS to remove large cell clumps and debris.</li>
</ol>
4. FACS analyses and sorting of human iPS cells-derived hepatocytes
<ol>
	<li>Set up the FACS machine for cell sorting following the manufacturer&#39;s instructions (see Table of Materials). 
	NOTE: Use an 85 &#181;m or 100 &#181;m nozzle size and an ND filter of 2.0 for hepatocytes. Use the FITC channel for detecting Alexa Flour 488 for ALCAM and the PE channel for detecting TMRM fluorescence for mitochondria.</li>
	<li>Adjust the FSC (forward scatter) and SSC (side scatter) voltages to properly visualize the cell population.</li>
	<li>Adjust the voltages of the FITC and PE fluorescent channels. Start with unlabeled cells and center the cell population at the bottom of each channel plot. Ensure labeled cells are appropriately positioned within the plot.</li>
	<li>Use a common gating strategy to eliminate doublets: Gate the major FSC-A vs. SSC-A population (Figure 2A), followed by FSC-H vs. -W, and then SSC-H vs. -W (Figure 2B,C).</li>
	<li>Gate the TMRMhi and ALCAM+ population (P2 in Figure 2E) to sort hepatocytes. Gate the TMRMhi and ALCAM- population (P1 in Figure 2E) to sort non-hepatocytes. Collect sorted hepatocytes and non-hepatocytes in 15 mL collection tubes containing hepatocyte maturation medium.</li>
	<li>Centrifuge the collection tube containing the sorted cells at 200 x g for 5 min at 18 &#176;C.</li>
	<li>Remove the supernatant and resuspend the cells in hepatocyte maturation medium.</li>
	<li>Seed the cells on mitomycin C-treated mouse embryonic fibroblasts (MEFs) or gel-like basement membrane matrix with hepatocyte maturation medium supplemented with 10 &#181;M Y-27632 and 20 nM ciclosporin A. Culture the cells in a 37 &#176;C CO2 incubator for 5 days.</li>
</ol>
5. Immunocytochemistry for detection of human iPS cells-derived hepatocytes
<ol>
	<li>Wash the cells cultured for 5 days after sorting with PBS (-).</li>
	<li>Fix the cells with 4% paraformaldehyde for 5 min at room temperature.</li>
	<li>Wash the cells with TBS-T buffer (1x TBS with 1% Tween 20) twice to remove the paraformaldehyde.</li>
	<li>Incubate the cells with 0.1% Triton X-100 diluted TBS-T buffer for 5 min at room temperature.</li>
	<li>Wash the cells with TBS-T buffer twice to remove Triton X-100.</li>
	<li>Add commercially available blocking solution (see Table of Materials) to the cells for 30 min at room temperature.</li>
	<li>Add primary antibodies against albumin (hepatocyte marker) and human nuclear antigen (hNA, see Table of Materials), diluted 1:50 in blocking solution.</li>
	<li>Incubate the cells at 4 &#176;C overnight. Wash the cells with TBS-T buffer twice.</li>
	<li>Add secondary antibodies: Alexa Fluor 488 donkey anti-rabbit IgG and Alexa Fluor 546 donkey anti-mouse IgG, diluted 1:100 in blocking solution (see Table of Materials).</li>
	<li>Incubate the cells at room temperature for 30 min. Wash the cells with TBS-T buffer twice.</li>
	<li>Evaluate hepatocyte purity by counting albumin-positive and hNA-positive cells under a fluorescence microscope. Calculate the percentage of albumin-positive cells per hNA-positive cell for both the P1 and P2 populations.</li>
</ol>

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

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balancing+serendipity+and+reproducibility:+Pluripotent+stem+cells+as+experimental+systems+for+intellectual+and+developmental+disorders.">Balancing serendipity and reproducibility: Pluripotent stem cells as experimental systems for intellectual and developmental disorders.</a> Stem Cell Reports. 16 (6), 1446-1457 (2021).</li><li>Chen, C. X. -. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardized+quality+control+workflow+to+evaluate+the+reproducibility+and+differentiation+potential+of+human+iPSCs+into+neurons.">Standardized quality control workflow to evaluate the reproducibility and differentiation potential of human iPSCs into neurons.</a> bioRxiv. , (2021).</li><li>Duinsbergen, D., Salvatori, D., Eriksson, M., Mikkers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumors+originating+from+induced+pluripotent+stem+cells+and+methods+for+their+prevention.">Tumors originating from induced pluripotent stem cells and methods for their prevention.</a> Annals of the New York Academy of Sciences. 1176, 197-204 (2009).</li><li>Nori, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+safety+issues+of+iPSC-based+cell+therapy+in+a+spinal+cord+injury+model:+oncogenic+transformation+with+epithelial-mesenchymal+transition.">Long-term safety issues of iPSC-based cell therapy in a spinal cord injury model: oncogenic transformation with epithelial-mesenchymal transition.</a> Stem Cell Reports. 4 (3), 360-373 (2015).</li><li>Hentze, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratoma+formation+by+human+embryonic+stem+cells:+evaluation+of+essential+parameters+for+future+safety+studies.">Teratoma formation by human embryonic stem cells: evaluation of essential parameters for future safety studies.</a> Stem Cell Research. 2 (3), 198-210 (2009).</li><li>Anderson, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+enrichment+of+cardiomyocytes+from+human+embryonic+stem+cells.">Transgenic enrichment of cardiomyocytes from human embryonic stem cells.</a> Molecular Therapy. 15 (11), 2027-2036 (2007).</li><li>Hidaka, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chamber-specific+differentiation+of+Nkx2.5-positive+cardiac+precursor+cells+from+murine+embryonic+stem+cells.">Chamber-specific differentiation of Nkx2.5-positive cardiac precursor cells from murine embryonic stem cells.</a> The FASEB Journal. 17 (6), 740-742 (2003).</li><li>Gassanov, N., Er, F., Zagidullin, N., Hoppe, U. 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The authors have nothing to disclose.

Due to their functions in nutrient metabolism and detoxification, hepatocytes possess a relatively large number of mitochondria compared to other cell types15. ALCAM is a member of the immunoglobulin superfamily and plays a role in cell adhesion and migration. It is expressed in various cell types, including hepatic, epithelial, lymphocytic, myeloid, fibroblast, and neuronal cells16. By utilizing a combination of the mitochondria-based method and the ALCAM antibody, human i...

Embryonic and induced pluripotent stem cells (ES and iPS, respectively) are considered promising cell sources for regenerative therapies. However, the efficiency of differentiating these cells into specific target cell types can vary, even when using the same cell line, protocol, and experimenter1,2,3,4. This variability may be attributed to the high sensitivity of human ES/iPS cells to their environment. Therefore, it is currently difficult to consistently obtain pure target cells. To achieve highly safe regenerative medicine, it is crucial to eliminate proliferative cells and undifferentiated stem cells in therapeutic cells, and advanced purification technology for target cells is essential5,6,7.
A cell sorter is a device that instantaneously analyzes individual cells and sorts live cells of interest based on the fluorescent signal strengths, offering a promising solution. This can be accomplished through antibody staining of cell type-specific surface markers or by utilizing cell type-specific reporter gene expressions. Using this technique, there are several reports on methods for purifying pluripotent stem cell-derived cardiomyocytes8,9,10 and hepatocytes11,12. Hattori et al. developed an innovative mitochondrial purification method using a cell sorter13. Taking advantage of the fact that cardiomyocytes have high energy demands through mitochondrial activity, staining the cells with the live mitochondria-indicative dye TMRM (tetramethylrhodamine methyl ester) can be used to label and highly purify cardiomyocytes by FACS from human ES cell-derived embryoid bodies containing various cell types. The absence of tumorigenicity was confirmed by teratoma formation assays with the purified cardiomyocytes. Furthermore, Yamashita et al. unexpectedly discovered a method to purify hepatocytes from human ES cell-derived embryoid bodies by isolating fractions with high mitochondrial activity and ALCAM-positive expression14. The rationale for this method is that hepatocytes also have a relatively high number of mitochondria due to their high consumption of ATP for nutrient metabolism and detoxification15, and hepatocytes express ALCAM, a member of the immunoglobulin superfamily, which plays a role in cell adhesion and migration16.
Previous highly purifying methods for pluripotent stem cell-derived hepatocytes required genetic modifications, and non-genetic purification methods had low efficiency17. The mitochondrial non-genetic method holds merit for achieving high purity. When hepatic progenitor cells are needed, CD133 and CD13- or Dlk1-based methods18,19 can be chosen. Although the accuracy of genome editing technology has advanced, the potential risk of unforeseen genomic changes (e.g., carcinogenesis) cannot be entirely eliminated. Methods based on mitochondrial activity, without involving genetic modification, can be free from such risks.
As a result of examining various mitochondrial indicators in neonatal rat cardiomyocytes, TMRM was observed to disappear completely within 24 h, whereas other dyes remained for at least 5 days13. Moreover, it is important to note that TMRM and JC-1 did not impact cell viability when assessed with the 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, while other dyes demonstrated varying effects on cell viability13. TMRM demonstrates higher safety.
To date, several two-dimensional (2D) differentiation methods have been developed as more efficient approaches for inducing differentiation compared to three-dimensional (3D) embryoid body formation. This is because step-wise differentiation-inducing compounds or cytokines can be uniformly administered to cells on a 2D plane, rather than in a 3D space. In this study, the previously reported method20 was modified to induce differentiation into human iPS cell-derived hepatocytes. Here, the details of the procedures for the up-to-date 2D differentiation and purification of hepatocytes derived from human iPS cells are described.

<table><tbody><tr><td>253G1 human iPS cell line</td><td>RIKEN BioResource Research Center</td><td>HPS0002</td><td></td></tr><tr><td>4% paraformaldehyde</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>163-20145</td><td></td></tr><tr><td>Activin A Solution, Human, Recombinant</td><td>NACALAI TESQUE, INC.</td><td>18585</td><td></td></tr><tr><td>Anti-Nuclei Antibody, clone 235-1</td><td>Chemicon</td><td>MAB1281</td><td>Antibody against human nuclear antigen</td></tr><tr><td>B-27 Supplement (50x), serum free</td><td>Thermo Fisher Scientific</td><td>17504044</td><td></td></tr><tr><td>BD FACSAria III</td><td>BD Biosciences</td><td></td><td>Cell sorter</td></tr><tr><td>CHIR-99021</td><td>MedChemExpress</td><td>HY-10182&amp;nbsp;</td><td></td></tr><tr><td>Ciclosporin A</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>035-18961</td><td></td></tr><tr><td>Collagenase</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>034-22363</td><td></td></tr><tr><td>Corning Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td><td>Corning</td><td>354230</td><td>Gel-like basement membrane matrix</td></tr><tr><td>CultureSure Y-27632</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>036-24023</td><td>ROCK inhibitor</td></tr><tr><td>Dexamethasone</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>047-18863</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>047-29353</td><td></td></tr><tr><td>Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-11055</td><td></td></tr><tr><td>Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546</td><td>Thermo Fisher Scientific</td><td>A10036</td><td></td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A-21206</td><td></td></tr><tr><td>Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap</td><td>Corning</td><td>352235</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Biowest</td><td>51820-500</td><td></td></tr><tr><td>GlutaMAX Supplement</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>Dipeptide L-Alanyl-L-Glutamine</td></tr><tr><td>Human/Mouse/Rat/Canine ALCAM/CD166 Antibody</td><td>R&amp;amp;D Systems</td><td>AF1172</td><td></td></tr><tr><td>Hydrocortisone 21-hemisuccinate sodium salt</td><td>Sigma-Aldrich</td><td>H2270</td><td></td></tr><tr><td>iMatrix-511 silk</td><td>Nippi</td><td>892021</td><td></td></tr><tr><td>ImmunoBlock</td><td>KAC</td><td>CTKN001</td><td>Blocking solution</td></tr><tr><td>ITS-G Supplement(&amp;times;100)</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>090-06741</td><td></td></tr><tr><td>Leibovitz&#039;s L-15 Medium</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>128-06075</td><td></td></tr><tr><td>N-Hexanoic-Try-Ile-(6)-amino Hexanoic amide (Dihexa)</td><td>Toronto Research Chemicals</td><td>H293745</td><td></td></tr><tr><td>Polyclonal Rabbit Anti-Human Albumin</td><td>Dako</td><td>A0001</td><td></td></tr><tr><td>Polyoxyethylene Sorbitan Monolaurate (Tween 20)</td><td>NACALAI TESQUE, INC.</td><td>28353-85</td><td></td></tr><tr><td>RPMI-1640</td><td>FUJIFILM Wako Pure Chemical Corporation</td><td>189-02025</td><td></td></tr><tr><td>Sodium L-Ascorbate</td><td>NACALAI TESQUE, INC.</td><td>03422-32</td><td></td></tr><tr><td>StemFit AK02N</td><td>REPROCELL</td><td>RCAK02N</td><td></td></tr><tr><td>TBS (10x)</td><td>NACALAI TESQUE, INC.</td><td>12748-31</td><td></td></tr><tr><td>Tetramethylrhodamine, methyl ester (TMRM)</td><td>Thermo Fisher Scientific</td><td>T668</td><td></td></tr><tr><td>Triton X-100</td><td>NACALAI TESQUE, INC.</td><td>28229-25</td><td></td></tr><tr><td>Trypan Blue Solution</td><td>NACALAI TESQUE, INC.</td><td>20577-34</td><td></td></tr><tr><td>TRYPSIN 250</td><td>Difco</td><td>215240</td><td></td></tr><tr><td>Tryptose phosphate broth solution</td><td>Sigma-Aldrich</td><td>T8159</td><td></td></tr></tbody></table>

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.

The timeline of the processes inducing human iPS cells to differentiate into hepatocytes through 2D culture (Figure 1A) and representative cell features (Figure 1B) are shown. Approximately on differentiation day 12, cells began to exhibit polygonal cell shapes and round nuclei, characteristic of hepatocytes. Some hepatocytes also displayed multinucleation.
FACS analysis was performed using the cells on differentiation day 27. To iden...

Watch this Scientific Journal Video about Application of Live Mitochondria Staining in Cell-Sorting to Purify Hepatocytes Derived from Human Induced Pluripotent Stem Cells at JoVE.com

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.

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 ...

application-live-mitochondria-staining-cell-sorting-to-purify

Research

JoVE Journal

Biology

434 Views.  Kansai Medical University. De reikwijdte van ons onderzoek is het zuiveren van door mensen geïnduceerde pluripotente stamcel-afgeleide hepatocyten, zonder genetische modificatie. We streven ernaar het probleem van het tumorigene risico aan te pakken dat gepaard gaat met de ongedifferentieerde cellen, onder degenen die gedifferentieerd zijn van menselijke iPS-cellen. Een van de huidige experimentele uitdagingen is een lage celzuiverheid, als gevolg van de mechanische opslag van het sorteren.We ontwikkelen een ni...

Video: Auteur in de schijnwerpers: Bevordering van hepatocytenzuivering uit door de mens geïnduceerde pluripotente stamcellen voor regeneratieve geneeskunde

Derivation of Highly Purified Cardiomyocytes from Human Induced Pluripotent Stem Cells Using Small Molecule-modulated Differentiation and Subsequent Glucose Starvation

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes available for basic research and translational applications. To differentiate hiPSCs into cardiomyocytes, various protocols including embryoid body (EB)-based differentiation and growth factor induction have been developed. However, these protocols are inefficient and highly variable in their ability to generate purified cardiomyocytes. Recently, a small molecule-based protocol utilizing modulation of Wnt/&#946;-Catenin signaling was shown to promote cardiac differentiation with high efficiency. With this protocol, greater than 50%-60% of differentiated cells were cardiac troponin-positive cardiomyocytes were consistently observed. To further increase cardiomyocyte purity, the differentiated cells were subjected to glucose starvation to specifically eliminate non-cardiomyocytes based on the metabolic differences between cardiomyocytes and non-cardiomyocytes. Using this selection strategy, we consistently obtained a greater than 30% increase in the ratio of cardiomyocytes to non-cardiomyocytes in a population of differentiated cells. These highly purified cardiomyocytes should enhance the reliability of results from human iPSC-based in vitro disease modeling studies and drug screening assays.

Here, we describe a robust protocol for human cardiomyocyte derivation that combines small molecule-modulated cardiac differentiation and glucose deprivation-mediated cardiomyocyte purification, enabling production of purified cardiomyocytes for the purposes of cardiovascular disease modeling and drug screening.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have become an important cell source to address the lack of primary cardiomyocytes ...

Developmental Biology

Stabilizing Hepatocellular Phenotype Using Optimized Synthetic Surfaces

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing and maintaining primary and pluripotent stem cell derived hepatocytes. While biological matrices are useful, they permit short term culture of hepatocytes, limiting their widespread application. We have attempted to overcome the limitations using a synthetic polymer coating. Polymers represent one of the broadest classes of biomaterials and possess a wide range of mechanical, physical and chemical properties, which can be fine-tuned for purpose. Importantly, such materials can be scaled to quality assured standards and display batch-to-batch consistency. This is essential if cells are to be expanded for high through-put screening in the pharmaceutical testing industry or for cellular based therapy. Polyurethanes (PUs) are one group of materials that have shown promise in cell culture. Our recent progress in optimizing a polyurethane coated surface, for long-term culture of human hepatocytes displaying stable phenotype, is presented and discussed.

This article will focus on developing polymer coated surfaces for long-term, stable culture of stem cell derived human hepatocytes.

Currently, one of the major limitations in cell biology is maintaining differentiated cell phenotype. Biological matrices are commonly used for culturing ...

Chemistry

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Biochemistry

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 ...

Electroporation-Mediated Delivery of Cas9 Ribonucleoproteins and mRNA into Freshly Isolated Primary Mouse Hepatocytes

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 as ribonucleoproteins (RNPs) and mRNA. Primary mouse hepatocytes were isolated using a three-step retrograde perfusion method resulting in high yields of up to 50 &#215; 106 cells per liver and cell viability of &#62;85%. This protocol provides detailed instructions for plating, staining, and culturing hepatocytes. The results indicate that electroporation provides a high transfection efficiency of 89%, as measured by the percentage of green fluorescent protein (GFP)-positive cells and modest cell viability of &#62;35% in mouse hepatocytes.
To demonstrate the utility of this approach, CRISPR-Cas9 targeting the hydroxyphenylpyruvate dioxygenase gene was electroporated into primary mouse hepatocytes as proof-of-principle gene editing to disrupt a therapeutic gene related to an inherited metabolic disease (IMD) of the liver. A higher on-target edit of 78% was observed for RNPs compared to 47% editing efficiency with mRNA. The functionality of hepatocytes was evaluated in vitro using an albumin assay that indicated that delivering CRISPR-Cas9 as RNPs and mRNA results in comparable cell viability in primary mouse hepatocytes. A promising application for this protocol is the generation of mouse models for human genetic diseases affecting the liver.

This protocol describes techniques for isolating primary mouse hepatocytes from the liver and electroporating CRISPR-Cas9 as ribonucleoproteins and mRNA to disrupt a therapeutic target gene associated with an inherited metabolic disease of the liver. The methods described result in high viability and high levels of gene modification after electroporation.

This protocol describes a fast and effective method for isolating primary mouse hepatocytes followed by electroporation-mediated delivery of CRISPR-Cas9 ...

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

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the &#34;gold standard&#34; for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine1. Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique.
Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation.
Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

A technique to isolate human hepatocytes and non-parenchymal liver cells from the same donor is described. The different liver cell types build the basis for functional liver models and tissue engineering. This new method aims to isolate liver cells in a high yield and viability.

Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic ...

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

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