Name: Author Spotlight: Advancing Hepatocyte Purification from Human Induced Pluripotent Stem Cells for Regenerative Medicine
Uploaded: 2023-12-01T12:50:00
Description: 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

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:...

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

<ol>
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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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelin+induces+differentiation+of+ANP-EGFP+expressing+embryonic+stem+cells+towards+a+pacemaker+phenotype.">Endothelin induces differentiation of ANP-EGFP expressing embryonic stem cells towards a pacemaker phenotype.</a> The FASEB Journal. 18 (14), 1710-1712 (2004).</li><li>Duan, Y., 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=Differentiation+and+enrichment+of+hepatocyte-like+cells+from+human+embryonic+stem+cells+in+vitro+and+in+vivo.">Differentiation and enrichment of hepatocyte-like cells from human embryonic stem cells in vitro and in vivo.</a> Stem Cells. 25 (12), 3058-3068 (2007).</li><li>Takayama, 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=Enrichment+of+high-functioning+human+iPS+cell-derived+hepatocyte-like+cells+for+pharmaceutical+research.">Enrichment of high-functioning human iPS cell-derived hepatocyte-like cells for pharmaceutical research.</a> Biomaterials. 161, 24-32 (2018).</li><li>Hattori, F., 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=Nongenetic+method+for+purifying+stem+cell-derived+cardiomyocytes.">Nongenetic method for purifying stem cell-derived cardiomyocytes.</a> Nature methods. 7, 61-66 (2009).</li><li>Yamashita, H., Fukuda, K., Hattori, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocyte-like+cells+derived+from+human+pluripotent+stem+cells+can+be+enriched+by+a+combination+of+mitochondrial+content+and+activated+leukocyte+cell+adhesion+molecule.">Hepatocyte-like cells derived from human pluripotent stem cells can be enriched by a combination of mitochondrial content and activated leukocyte cell adhesion molecule.</a> JMA Journal. 2 (2), 174-183 (2019).</li><li>Fernandez-Vizarra, E., Enr&#237;quez, J., P&#233;rez-Martos, A., Montoya, J., Fern&#225;ndez-Silva, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+differences+in+mitochondrial+activity+and+biogenesis.">Tissue-specific differences in mitochondrial activity and biogenesis.</a> Mitochondrion. 11, 207-213 (2010).</li><li>Swart, G. W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+leukocyte+cell+adhesion+molecule+(CD166/ALCAM):+Developmental+and+mechanistic+aspects+of+cell+clustering+and+cell+migration.">Activated leukocyte cell adhesion molecule (CD166/ALCAM): Developmental and mechanistic aspects of cell clustering and cell migration.</a> European Journal of Cell Biology. 81 (6), 313-321 (2002).</li><li>Peters, D. T., 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=Asialoglycoprotein+receptor+1+is+a+specific+cell-surface+marker+for+isolating+hepatocytes+derived+from+human+pluripotent+stem+cells.">Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells.</a> Development. 143 (9), 1475-1481 (2016).</li><li>Kamiya, A., Inagaki, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+and+progenitor+cell+systems+in+liver+development+and+regeneration.">Stem and progenitor cell systems in liver development and regeneration.</a> Hepatology Research. 45 (1), 29-37 (2015).</li><li>Yanagida, A., Ito, K., Chikada, H., Nakauchi, H., Kamiya, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+In+vitro+expansion+system+for+generation+of+human+ips+cell-derived+hepatic+progenitor-like+cells+exhibiting+a+bipotent+differentiation+potential.">An In vitro expansion system for generation of human ips cell-derived hepatic progenitor-like cells exhibiting a bipotent differentiation potential.</a> PLOS One. 8 (7), e67541 (2013).</li><li>Siller, R., Greenhough, S., Naumovska, E., Sullivan, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule-driven+hepatocyte+differentiation+of+human+pluripotent+stem+cells.">Small-molecule-driven hepatocyte differentiation of human pluripotent stem cells.</a> Stem Cell Reports. 4 (5), 939-952 (2015).</li><li>Hirata, 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=ALCAM+(CD166)+is+a+surface+marker+for+early+murine+cardiomyocytes.">ALCAM (CD166) is a surface marker for early murine cardiomyocytes.</a> Cells, Tissues, Organs. 184 (3-4), 172-180 (2006).</li><li>Tohyama, 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=Distinct+metabolic+flow+enables+large-scale+purification+of+mouse+and+human+pluripotent+stem+cell-derived+cardiomyocytes.">Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes.</a> Cell Stem Cell. 12 (1), 127-137 (2013).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#160;</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&#38;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(&#215;100)</td>
			<td>FUJIFILM Wako Pure Chemical Corporation</td>
			<td>090-06741</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovitz&#39;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.

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

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

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

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 ...

The scope of our research is to purify human induced pluripotent stem cell-derived hepatocytes, using without genetic modification. We aim to address the issue of the tumorigenic risk associated with the undifferentiated cells, among those differentiated from human iPS cells. One of the current experimental challenges is a low cell purity, due to the mechanical storage of sorting.We are developing a new purification method, that utilizes the unique metabolism features of hepatocytes, for large-scale purification. One non-cell sorting method for hepatic progenitor cells cannot obtain pure hepatocytes. Another one for mature hepatocytes can only treat a small fraction of hepatocytes, because of the limited expression of the marker protein.In contrast, our approach can obtain hepatocytes at different maturation levels with a higher eve. Human iPS cell-derived hepatocytes are extremely immature, and show more reduced hepatocyte macro expression after purification. Our future goal is to enhance hepatocyte modulation using organoidal technology and day-to-day advancements to regenerative medicine and drug discovery.