Name: Autore in primo piano: Avanzamento della purificazione degli epatociti da cellule staminali pluripotenti indotte umane per la medicina rigenerativa
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

Questo lavoro è stato sostenuto dal Ministero dell'Istruzione, della Cultura, dello Sport, della Scienza e della Tecnologia numero di sovvenzione [23390072].

Questo studio ha utilizzato cellule iPS umane ottenute commercialmente (ceppo 253G1) (vedi Tabella dei materiali).
1. Mantenimento delle cellule iPS umane
<ol><li>Mantenere le cellule iPS umane in condizioni prive di alimentatore in terreno AK02 su piastre di coltura rivestite con iMatrix-511 da 0,25 μg/cm2 (vedere la tabella dei materiali).</li>
</ol>2. Differenziamento epatico di cellule iPS umane ...

Purificazione di epatociti umani derivati da iPSC mediante smistamento mitocondriale-ALCAM

<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>

A causa delle loro funzioni nel metabolismo dei nutrienti e nella disintossicazione, gli epatociti possiedono un numero relativamente elevato di mitocondri rispetto ad altri tipi di cellule15. ALCAM è un membro della superfamiglia delle immunoglobuline e svolge un ruolo nell'adesione e nella migrazione cellulare. È espresso in vari tipi di cellule, tra cui cellule epatiche, epiteliali, linfocitiche, mieloidi, fibroblasti e neuronali16. Utilizzando una combinazione del met...

Le cellule staminali embrionali e pluripotenti indotte (ES e iPS, rispettivamente) sono considerate fonti cellulari promettenti per le terapie rigenerative. Tuttavia, l'efficienza della differenziazione di queste cellule in specifici tipi di cellule bersaglio può variare, anche quando si utilizza la stessa linea cellulare, lo stesso protocollo e lo stesso sperimentatore 1,2,3,4. Questa variabilità può essere attribuita all'elevata sensibilità delle cellule ES/iPS umane al loro ambiente. Pertanto, attualmente è difficile ottenere costantemente cellule bersaglio pure. Per ottenere una medicina rigenerativa altamente sicura, è fondamentale eliminare le cellule proliferative e le cellule staminali indifferenziate nelle cellule terapeutiche ed è essenziale una tecnologia avanzata di purificazione per le cellule bersaglio 5,6,7.
Un selezionatore di cellule è un dispositivo che analizza istantaneamente le singole cellule e ordina le cellule vive di interesse in base all'intensità del segnale fluorescente, offrendo una soluzione promettente. Ciò può essere ottenuto attraverso la colorazione con anticorpi di marcatori di superficie specifici per il tipo di cellula o utilizzando espressioni geniche reporter specifiche per il tipo di cellula. Utilizzando questa tecnica, ci sono diversi rapporti su metodi per purificare i cardiomiociti 8,9,10 e gli epatociti11,12 derivati da cellule staminali pluripotenti. Hattori et al. hanno sviluppato un innovativo metodo di purificazione mitocondriale utilizzando un selezionatore di cellule13. Sfruttando il fatto che i cardiomiociti hanno un'elevata richiesta di energia attraverso l'attività mitocondriale, la colorazione delle cellule con il colorante mitocondriale vivo TMRM (estere metilmetilico-rodamina) può essere utilizzato per marcare e purificare altamente i cardiomiociti mediante FACS da corpi embrioidi derivati da cellule ES umane contenenti vari tipi di cellule. L'assenza di tumorigenicità è stata confermata da saggi di formazione di teratomi con i cardiomiociti purificati. Inoltre, Yamashita et al. hanno inaspettatamente scoperto un metodo per purificare gli epatociti da corpi embrioidi derivati da cellule ES umane isolando frazioni con elevata attività mitocondriale ed espressione ALCAM-positiva14. Il razionale di questo metodo è che gli epatociti hanno anche un numero relativamente alto di mitocondri a causa del loro elevato consumo di ATP per il metabolismo dei nutrienti e la disintossicazione15, e gli epatociti esprimono ALCAM, un membro della superfamiglia delle immunoglobuline, che svolge un ruolo nell'adesione e nella migrazione cellulare16.
I precedenti metodi altamente purificanti per gli epatociti derivati da cellule staminali pluripotenti richiedevano modificazioni genetiche e i metodi di purificazione non genetici avevano una bassa efficienza17. Il metodo non genetico mitocondriale ha il merito di raggiungere un'elevata purezza. Quando sono necessarie cellule progenitrici epatiche, è possibile scegliere i metodi CD133 e CD13 o Dlk1 18,19. Sebbene l'accuratezza della tecnologia di editing del genoma sia avanzata, il potenziale rischio di cambiamenti genomici imprevisti (ad esempio, la cancerogenesi) non può essere completamente eliminato. I metodi basati sull'attività mitocondriale, senza comportare modificazioni genetiche, possono essere esenti da tali rischi.
Come risultato dell'esame di vari indicatori mitocondriali nei cardiomiociti neonatali di ratto, è stato osservato che il TMRM scompare completamente entro 24 ore, mentre altri coloranti sono rimasti per almeno 5 giorni13. Inoltre, è importante notare che TMRM e JC-1 non hanno influenzato la vitalità cellulare quando valutati con il test 3-(4,5-dimetil-tiazol-2-il)-2,5-difeniltetrazolio bromuro (MTT), mentre altri coloranti hanno dimostrato effetti variabili sulla vitalità cellulare13. TMRM dimostra una maggiore sicurezza.
Ad oggi, sono stati sviluppati diversi metodi di differenziazione bidimensionale (2D) come approcci più efficienti per indurre la differenziazione rispetto alla formazione di corpi embrioidi tridimensionali (3D). Questo perché i composti o le citochine che inducono la differenziazione graduale possono essere somministrati uniformemente alle cellule su un piano 2D, piuttosto che in uno spazio 3D. In questo studio, il metodo20 precedentemente riportato è stato modificato per indurre la differenziazione in epatociti derivati da cellule iPS umane. Qui vengono descritti i dettagli delle procedure per la differenziazione e la purificazione 2D aggiornate di epatociti derivati da cellule iPS umane.

<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

Le cellule staminali embrionali umane (ES) e le cellule staminali pluripotenti indotte (iPS) hanno potenziali applicazioni nella medicina rigenerativa basata sulle cellule per il trattamento di organi gravemente malati grazie alla loro proliferazione illimitata e alle loro proprietà pluripotenti. Tuttavia, la differenziazione delle cellule ES/iPS umane in tipi di cellule bersaglio pure al 100% è impegnativa a causa della loro elevata sensibilità all'ambiente. La tumorigenesi dopo il trapianto è causata da cellule contaminate, proliferanti e indifferenziate, rendendo la tecnologia ad alta purificazione essenziale per la realizzazione sicura della medicina rigenerativa. Per mitigare il rischio di tumorigenesi, è stata sviluppata una tecnologia ad alta purificazione per gli epatociti derivati da cellule iPS umane. Il metodo utilizza FACS (fluorescence-activated cell sorting) utilizzando una combinazione di alto contenuto mitocondriale e il marcatore di superficie cellulare ALCAM (activated leukocyte cell adhesion molecule) senza modificazioni genetiche. Il 97% ± lo 0,38% (n = 5) degli epatociti purificati utilizzando questo metodo hanno mostrato espressione proteica di albumina. Questo articolo si propone di fornire procedure dettagliate per questo metodo, come applicato al più recente metodo di differenziazione bidimensionale per cellule iPS umane in epatociti.

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.

Autore in primo piano: Avanzamento della purificazione degli epatociti da cellule staminali pluripotenti indotte umane per la medicina rigenerativa

Applicazione della colorazione dei mitocondri vivi nella selezione cellulare per purificare gli epatociti derivati da cellule staminali pluripotenti indotte umane

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.

Viene mostrata la cronologia dei processi che inducono le cellule iPS umane a differenziarsi in epatociti attraverso la coltura 2D (Figura 1A) e le caratteristiche cellulari rappresentative (Figura 1B). Approssimativamente il 12° giorno di differenziazione, le cellule hanno iniziato a mostrare forme cellulari poligonali e nuclei rotondi, caratteristici degli epatociti. Alcuni epatociti mostravano anche multinucleazione.
L'analisi FAC...

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

Gli epatociti derivati da cellule staminali pluripotenti possono essere purificati attraverso la selezione cellulare, utilizzando una combinazione di colorazione con molecole di adesione alle cellule mitocondriali e leucocitarie attivate (ALCAM, nota anche come CD166).

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

Lo scopo della nostra ricerca è quello di purificare gli epatociti derivati da cellule staminali pluripotenti indotte umane, utilizzando senza modificazioni genetiche. Ci proponiamo di affrontare il problema del rischio tumorigenico associato alle cellule indifferenziate, tra quelle differenziate dalle cellule iPS umane. Una delle attuali sfide sperimentali è la bassa purezza delle celle, dovuta allo stoccaggio meccanico della cernita.Stiamo sviluppando un nuovo metodo di purificazione, che utilizza le caratteristiche uniche del metabolismo degli epatociti, per una purificazione su larga scala. Un metodo di selezione non cellulare per le cellule progenitrici epatiche non può ottenere epatociti puri. Un altro per gli epatociti maturi può trattare solo una piccola frazione di epatociti, a causa della limitata espressione della proteina marcatrice.Al contrario, il nostro approccio può ottenere epatociti a diversi livelli di maturazione con un'età più alta. Gli epatociti derivati da cellule iPS umane sono estremamente immaturi e mostrano un'espressione più ridotta delle macro epatocitarie dopo la purificazione. Il nostro obiettivo futuro è quello di migliorare la modulazione degli epatociti utilizzando la tecnologia organoidale e i progressi quotidiani verso la medicina rigenerativa e la scoperta di farmaci.

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.

Ricerca

Biologia

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.

Differenziazione epatica di cellule staminali pluripotenti indotte dall'uomo in colture 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.

Preparazione cellulare per la purificazione di epatociti derivati da cellule staminali pluripotenti indotte dall'uomo

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.