Name: Pleins feux sur l’auteur : Faire progresser la purification des hépatocytes à partir de cellules souches pluripotentes induites par l’homme pour la médecine régénérative
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

Ce travail a été soutenu par le ministère de l’Éducation, de la Culture, des Sports, de la Science et de la Technologie n° de subvention [23390072].

Cette étude a utilisé des cellules iPS humaines obtenues commercialement (souche 253G1) (voir le tableau des matériaux).
1. Maintenance des cellules iPS humaines
<ol><li>Maintenir les cellules iPS humaines dans des conditions exemptes de feeder dans un milieu AK02 sur des boîtes de culture recouvertes de 0,25 μg/cm2 iMatrix-511 (voir le tableau des matériaux).</li>
</ol>2. Différenciation hépa...

Purification d’hépatocytes humains dérivés d’iPSC par tri mitochondrial-ALCAM

<ol>
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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. 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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>

En raison de leurs fonctions dans le métabolisme des nutriments et la détoxification, les hépatocytes possèdent un nombre relativement important de mitochondries par rapport aux autres types de cellules15. ALCAM fait partie de la superfamille des immunoglobulines et joue un rôle dans l’adhésion et la migration cellulaires. Il est exprimé dans divers types de cellules, notamment les cellules hépatiques, épithéliales, lymphoïdes, myéloïdes, fibroblastiques et neuronales<sup class="xre...

Les cellules souches embryonnaires et pluripotentes induites (ES et iPS, respectivement) sont considérées comme des sources cellulaires prometteuses pour les thérapies régénératives. Cependant, l’efficacité de la différenciation de ces cellules en types de cellules cibles spécifiques peut varier, même en utilisant la même lignée cellulaire, le même protocole et le même expérimentateur 1,2,3,4. Cette variabilité peut être attribuée à la grande sensibilité des cellules ES/iPS humaines à leur environnement. Par conséquent, il est actuellement difficile d’obtenir de manière cohérente des cellules cibles pures. Pour parvenir à une médecine régénérative hautement sûre, il est crucial d’éliminer les cellules prolifératives et les cellules souches indifférenciées dans les cellules thérapeutiques, et une technologie de purification avancée pour les cellules cibles est essentielle 5,6,7.
Un trieur de cellules est un appareil qui analyse instantanément les cellules individuelles et trie les cellules vivantes d’intérêt en fonction de l’intensité du signal fluorescent, offrant ainsi une solution prometteuse. Cela peut être accompli par la coloration par anticorps de marqueurs de surface spécifiques au type cellulaire ou en utilisant des expressions de gènes rapporteurs spécifiques au type cellulaire. En utilisant cette technique, il existe plusieurs rapports sur les méthodes de purification des cardiomyocytes 8,9,10 et des hépatocytes11,12 dérivés de cellules souches pluripotentes. Hattori et al. ont développé une méthode innovante de purification mitochondriale à l’aide d’un trieur de cellules13. Tirant parti du fait que les cardiomyocytes ont des besoins énergétiques élevés par le biais de l’activité mitochondriale, la coloration des cellules avec le colorant TMRM (ester méthylique de tétraméthylrhodamine) peut être utilisée pour marquer et purifier fortement les cardiomyocytes par FACS à partir de corps embryoïdes dérivés de cellules ES humaines contenant divers types de cellules. L’absence de tumorigénicité a été confirmée par des tests de formation de tératomes avec les cardiomyocytes purifiés. De plus, Yamashita et al. ont découvert de manière inattendue une méthode pour purifier les hépatocytes des corps embryoïdes dérivés de cellules ES humaines en isolant des fractions à forte activité mitochondriale et à expression ALCAM-positive14. La raison de cette méthode est que les hépatocytes ont également un nombre relativement élevé de mitochondries en raison de leur forte consommation d’ATP pour le métabolisme des nutriments et la détoxification15, et les hépatocytes expriment ALCAM, un membre de la superfamille des immunoglobulines, qui joue un rôle dans l’adhésion et la migration cellulaires16.
Les méthodes précédentes de purification élevée pour les hépatocytes dérivés de cellules souches pluripotentes nécessitaient des modifications génétiques, et les méthodes de purification non génétiques avaient une faible efficacité17. La méthode mitochondriale non génétique a le mérite d’atteindre une grande pureté. Lorsque des cellules progénitrices hépatiques sont nécessaires, il est possible de choisir des méthodes basées sur CD133 et CD13 ou Dlk118,19. Bien que la précision de la technologie d’édition du génome ait progressé, le risque potentiel de changements génomiques imprévus (par exemple, la cancérogenèse) ne peut pas être entièrement éliminé. Les méthodes basées sur l’activité mitochondriale, sans impliquer de modification génétique, peuvent être exemptes de tels risques.
À la suite de l’examen de divers indicateurs mitochondriaux dans les cardiomyocytes néonatals de rats, on a observé que la TMRM disparaissait complètement en 24 heures, tandis que les autres colorants restaient pendant au moins 5 jours13. De plus, il est important de noter que TMRM et JC-1 n’ont pas eu d’impact sur la viabilité cellulaire lorsqu’ils ont été évalués à l’aide de l’essai au bromure de 3-(4,5-diméthyl-thiazol-2-yl)-2,5-diphényltétrazolium (MTT), tandis que d’autres colorants ont démontré des effets variables sur la viabilité cellulaire13. TMRM démontre une sécurité accrue.
À ce jour, plusieurs méthodes de différenciation bidimensionnelle (2D) ont été développées comme approches plus efficaces pour induire la différenciation par rapport à la formation de corps embryoïdes tridimensionnels (3D). En effet, les composés ou cytokines induisant la différenciation par étapes peuvent être administrés uniformément aux cellules sur un plan 2D, plutôt que dans un espace 3D. Dans cette étude, la méthode20 précédemment rapportée a été modifiée pour induire la différenciation en hépatocytes dérivés de cellules iPS humaines. Nous décrirons ici les détails des procédures de différenciation et de purification 2D modernes des hépatocytes dérivés de cellules iPS humaines.

<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

Les cellules souches embryonnaires humaines (SE) et les cellules souches pluripotentes induites (iPS) ont des applications potentielles en médecine régénérative cellulaire pour traiter les organes gravement malades en raison de leur prolifération illimitée et de leurs propriétés pluripotentes. Cependant, il est difficile de différencier les cellules ES/iPS humaines en types de cellules cibles 100 % pures en raison de leur grande sensibilité à l’environnement. La tumorigenèse après transplantation est causée par des cellules contaminées, proliférantes et indifférenciées, ce qui rend la technologie de haute purification essentielle pour la réalisation en toute sécurité de la médecine régénérative. Pour atténuer le risque de tumorigenèse, une technologie de purification élevée a été développée pour les hépatocytes dérivés de cellules iPS humaines. La méthode utilise le FACS (tri cellulaire activé par fluorescence) en utilisant une combinaison de contenu mitochondrial élevé et du marqueur de surface cellulaire ALCAM (molécule d’adhésion cellulaire leucocytaire activée) sans modification génétique. 97 % ± 0,38 % (n = 5) des hépatocytes purifiés utilisant cette méthode présentaient une expression protéique de l’albumine. Cet article vise à fournir des procédures détaillées pour cette méthode, telle qu’appliquée à la méthode de différenciation bidimensionnelle la plus récente pour les cellules iPS humaines en hépatocytes.

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.

Pleins feux sur l’auteur : Faire progresser la purification des hépatocytes à partir de cellules souches pluripotentes induites par l’homme pour la médecine régénérative

Application de la coloration des mitochondries vivantes dans le tri cellulaire pour purifier les hépatocytes dérivés de cellules souches pluripotentes induites par l’homme

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.

La chronologie des processus induisant les cellules iPS humaines à se différencier en hépatocytes par culture 2D (Figure 1A) et les caractéristiques cellulaires représentatives (Figure 1B) sont présentées. Approximativement au 12e jour de différenciation, les cellules ont commencé à présenter des formes cellulaires polygonales et des noyaux ronds, caractéristiques des hépatocytes. Certains hépatocytes ont également montré une multinucléation.</p...

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

Les hépatocytes dérivés de cellules souches pluripotentes peuvent être purifiés par tri cellulaire, en utilisant une combinaison de coloration de la molécule d’adhésion des cellules leucocytaires activées (ALCAM, également connue sous le nom de CD166).

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

Le champ d’application de notre recherche est de purifier les hépatocytes dérivés de cellules souches pluripotentes induites par l’homme, en utilisant sans modification génétique. Nous visons à aborder la question du risque tumorigène associé aux cellules indifférenciées, parmi celles différenciées des cellules iPS humaines. L’un des défis expérimentaux actuels est la faible pureté des cellules, en raison du stockage mécanique du tri.Nous développons une nouvelle méthode de purification, qui utilise les caractéristiques métaboliques uniques des hépatocytes, pour une purification à grande échelle. Une méthode de tri non cellulaire pour les cellules progénitrices hépatiques ne peut pas obtenir d’hépatocytes purs. Un autre pour les hépatocytes matures ne peut traiter qu’une petite fraction des hépatocytes, en raison de l’expression limitée de la protéine marqueur.En revanche, notre approche permet d’obtenir des hépatocytes à différents niveaux de maturation avec une eve plus élevée. Les hépatocytes humains dérivés de cellules iPS sont extrêmement immatures et présentent une expression plus réduite des macro-hépatocytes après purification. Notre objectif futur est d’améliorer la modulation des hépatocytes à l’aide de la technologie organoïdale et des progrès quotidiens de la médecine régénérative et de la découverte de médicaments.

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

Research

JoVE Journal

Biology

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.

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.

Recherche

Biologie

Hepatic Differentiation of Human-Induced Pluripotent Stem Cells in 2D Cultures

To begin, coat each well of a six-well plate with one milliliter of basement membrane matrix and incubate at 37 degrees Celsius for one hour. Remove the coated matrix from the plate and add human-induced pluripotent stem cells prepared in AK02N medium supplemented with 10 micromolar ROCK inhibitor. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for three days.After three days, on day zero of the initiating hepatic differentiation, wash the cells once with RPMI 1640 medium. Next, to initiate endoderm differentiation, add RPMI B27 GlutaMAX supplemented with three to six micromolar CHIR 99021 and 100 nanograms per milliliter Activin A into the plate. Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours.On day one, replace the existing medium with RPMI B27 GlutaMAX supplemented with 50 nanograms per milliliter of Activin A.Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 24 hours. On day two, to induce hepatic progenitor differentiation, replace the existing medium with RPMI B27 GlutaMAX medium supplemented with 1%dimethyl sulfoxide, and continue incubation for up to five days. On day seven, to initiate hepatocyte maturation, switch to hepatocyte maturation medium and incubate for 20 days at 37 degrees Celsius and 5%carbon dioxide.On day 27, human-induced pluripotent stem cells displayed polygonal cell shapes and rounded nuclei, characteristics of hepatocytes.

Différenciation hépatique de cellules souches pluripotentes induites par l’homme dans des cultures 2D

Cell Preparation for Purification of Human-Induced Pluripotent Stem Cells-Derived Hepatocytes

After 27 days of initiating hepatic differentiation of human induced pluripotent stem cells, wash the cells with two milliliters of PBS per well. Prepare the enzyme solution in ADS buffer using the given components. Add one milliliter of solution per well of a six well plate.Place the plate at 37 degrees Celsius on a rotating shaker for about two hours to detach the cells from the plate. Confirm the cell detachment under a microscope, then pipette the cell suspension to disperse into single cells, and collect them in a 50 milliliter tube containing the hepatocyte maturation medium. Centrifuge the cell suspension at 200G for five minutes at 18 degrees Celsius.And using an aspirator, remove the supernatant. To stain the mitochondria of cells, resuspend the pellet in five milliliters of hepatocyte maturation medium containing 100 nanomolar TMRM. Incubate the cells for 30 minutes at 37 degrees Celsius while protecting them from light.Again, centrifuge the cell suspension and resuspend the pellet in one to five milliliters of cold ADS buffer containing 2%FBS. After cell counting, aliquot 500, 000 cells to a 1.5 milliliter tube and label it as the no primary antibody control. Centrifuge the remaining cell suspension at 200G for five minutes at four degrees Celsius, and remove the supernatant.Add 100 microliters of diluted primary antibody per one million cells. Transfer the cell suspension to a 1.5 milliliter tube and incubate on ice for 50 minutes. After incubation, centrifuge the cell suspension and wash the cells twice with cold ADS buffer containing 2%FBS.Now add 100 microliters of diluted secondary antibody per one million cells to the primary antibody, stained or unstained cells, and incubate for 30 minutes on ice. Again, centrifuge the cell suspension and wash the cells twice with cold ADS buffer containing 2%FBS. After resuspending the cells in ADS buffer, filter them through a snap cap cell strainer and place the tube on ice.

PRÉPARATION CELLULAIRE POUR LA PURIFICATION D’HÉPATOCYTES DÉRIVÉS DE CELLULES SOUCHES PLURIPOTENTES INDUITES PAR L’HOMME

Fluorescence-Activated Cell Sorting of Hepatocytes Derived from Human Induced Pluripotent Stem Cells

To begin, set up the fluorescence-activated cell sorting, or FACS machine, for cell sorting following the manufacturer's instructions. Place the sample tube on the FACS machine and load it. Gate TMRM-high and ALCAM negative population in the P1 region to sort non-hepatocytes.Then gate the TMRM high and ALCAM positive population in the P2 region to sort hepatocytes. Collect sorted cells in 15-milliliter collection tubes containing hepatocyte maturation medium. After sorting, remove the collection tube from the FACS machine.Centrifuge the sorted cell suspension at 200 G for five minutes at 18 degrees Celsius. Remove the supernatant, and re-suspend the pellet in two milliliters of hepatocyte maturation medium with 10 micromolar rock inhibitor and 20 nanomolar cyclosporine A.Seed the cells on mitomycin C-treated mouse embryonic fibroblasts with hepatocyte maturation medium in a six-well plate. Culture the cells in a 37-degree Celsius carbon dioxide incubator for five days before immuno staining for hepatocyte purity test.FACS analysis conducted on day 27 of hepatic differentiation revealed TMRM high and ALCAM positive population. The immunohistochemical staining of P1 and P2 cells cultured on mouse embryonic fibroblasts after sorting is shown. A purity test by counting albumin-positive cells showed 0%of P1, and approximately 97%of P2 cells were hepatocyte-like cells.