Name: Yazar Spotlight: Rejeneratif Tıp için İnsan Kaynaklı Pluripotent Kök Hücrelerden Hepatosit Saflaştırmasının İlerletilmesi
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

Bu çalışma Milli Eğitim, Kültür, Spor, Bilim ve Teknoloji Bakanlığı hibe numarası [23390072] tarafından desteklenmiştir.

Bu çalışmada ticari olarak elde edilen insan iPS hücreleri (253G1 suşu) kullanılmıştır (bkz.
1. İnsan iPS hücrelerinin bakımı
<ol><li>İnsan iPS hücrelerini, 0,25 μg/cm2 iMatrix-511 ile kaplanmış kültür kaplarında AK02 ortamında besleyici içermeyen koşullarda tutun (bkz.</li>
</ol>2. 2D kültürlerde insan iPS hücrelerinin hepatik farklılaşması
<p class="jove_co...

İnsan iPSC'den Elde Edilen Hepatositlerin Mitokondriyal-ALCAM Sınıflandırması ile Saflaştırılması

<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. 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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>

Besin metabolizması ve detoksifikasyondaki işlevleri nedeniyle, hepatositler diğer hücre tiplerine kıyasla nispeten daha fazla sayıda mitokondriye sahiptir15. ALCAM, immünoglobulin süper ailesinin bir üyesidir ve hücre yapışması ve göçünde rol oynar. Hepatik, epitelyal, lenfositik, miyeloid, fibroblast ve nöronal hücreler dahil olmak üzere çeşitli hücre tiplerinde eksprese edilir16. Mitokondri bazlı yöntem ve ALCAM antikorunun bir kombinasyonu kullan...

Embriyonik ve indüklenmiş pluripotent kök hücreler (sırasıyla ES ve iPS), rejeneratif tedaviler için umut verici hücre kaynakları olarak kabul edilir. Bununla birlikte, bu hücreleri belirli hedef hücre tiplerine ayırmanın verimliliği, aynı hücre hattını, protokolü ve deneyci 1,2,3,4 kullanıldığında bile değişebilir. Bu değişkenlik, insan ES/iPS hücrelerinin çevrelerine karşı yüksek hassasiyetine bağlanabilir. Bu nedenle, şu anda tutarlı bir şekilde saf hedef hücreler elde etmek zordur. Son derece güvenli rejeneratif tıp elde etmek için, terapötik hücrelerde proliferatif hücreleri ve farklılaşmamış kök hücreleri ortadan kaldırmak çok önemlidir ve hedef hücreler için gelişmiş saflaştırma teknolojisi esastır 5,6,7.
Hücre sıralayıcı, tek tek hücreleri anında analiz eden ve floresan sinyal güçlerine dayalı olarak ilgilenilen canlı hücreleri sıralayan ve umut verici bir çözüm sunan bir cihazdır. Bu, hücre tipine özgü yüzey belirteçlerinin antikor boyaması yoluyla veya hücre tipine özgü raportör gen ekspresyonları kullanılarak gerçekleştirilebilir. Bu tekniği kullanarak, pluripotent kök hücre kaynaklı kardiyomiyositler 8,9,10 ve hepatositler 11,12'nin saflaştırılması yöntemleri hakkında birkaç rapor vardır. Hattori ve ark. bir hücre sıralayıcı13 kullanarak yenilikçi bir mitokondriyal saflaştırma yöntemi geliştirdi. Kardiyomiyositlerin mitokondriyal aktivite yoluyla yüksek enerji taleplerine sahip olduğu gerçeğinden yararlanarak, hücrelerin canlı mitokondri gösterge boyası TMRM (tetrametilrodamin metil ester) ile boyanması, çeşitli hücre tipleri içeren insan ES hücresinden türetilmiş embriyoid cisimlerinden FACS ile kardiyomiyositleri etiketlemek ve yüksek oranda saflaştırmak için kullanılabilir. Tümörjenitenin yokluğu, saflaştırılmış kardiyomiyositlerle teratom oluşum testleri ile doğrulandı. Ayrıca, Yamashita ve ark. beklenmedik bir şekilde, yüksek mitokondriyal aktiviteye ve ALCAM pozitif ekspresyona sahip fraksiyonları izole ederek insan ES hücresinden türetilmiş embriyoid cisimlerinden hepatositleri saflaştırmak için bir yöntem keşfettiler14. Bu yöntemin mantığı, hepatositlerin besin metabolizması ve detoksifikasyon için yüksek ATP tüketimleri nedeniyle nispeten yüksek sayıda mitokondriye sahip olmaları15 ve hepatositlerin, hücre yapışması ve göçünde rol oynayan immünoglobulin süper ailesinin bir üyesi olan ALCAM'ı eksprese etmesidir16.
Pluripotent kök hücre türevli hepatositler için önceki yüksek saflaştırma yöntemleri genetik modifikasyonlar gerektiriyordu ve genetik olmayan saflaştırma yöntemleri düşük verimliliğe sahipti17. Mitokondriyal genetik olmayan yöntem, yüksek saflık elde etmek için değer taşır. Hepatik progenitör hücrelere ihtiyaç duyulduğunda CD133 ve CD13 veya Dlk1 tabanlı yöntemler18,19 seçilebilir. Genom düzenleme teknolojisinin doğruluğu ilerlemiş olsa da, öngörülemeyen genomik değişikliklerin (örneğin, karsinogenez) potansiyel riski tamamen ortadan kaldırılamaz. Genetik modifikasyon içermeden mitokondriyal aktiviteye dayalı yöntemler bu tür risklerden muaf olabilir.
Yenidoğan sıçan kardiyomiyositlerinde çeşitli mitokondriyal indikatörlerin incelenmesi sonucunda, TMRM'nin 24 saat içinde tamamen kaybolduğu, diğer boyaların ise en az 5 gün kaldığı gözlenmiştir13. Ayrıca, TMRM ve JC-1'in 3- (4,5-dimetil-tiyazol-2-il) -2,5-difeniltetrazolyum bromür (MTT) testi ile değerlendirildiğinde hücre canlılığını etkilemediğini, diğer boyaların ise hücre canlılığı üzerinde farklı etkiler gösterdiğini belirtmek önemlidir13. TMRM daha yüksek güvenlik gösterir.
Bugüne kadar, üç boyutlu (3D) embriyoid cisim oluşumuna kıyasla farklılaşmayı indüklemek için daha etkili yaklaşımlar olarak birkaç iki boyutlu (2D) farklılaşma yöntemi geliştirilmiştir. Bunun nedeni, kademeli farklılaşmayı indükleyen bileşiklerin veya sitokinlerin, hücrelere 3 boyutlu bir alanda değil, 2 boyutlu bir düzlemde eşit şekilde uygulanabilmesidir. Bu çalışmada, daha önce bildirilen yöntem20 , insan iPS hücresinden türetilmiş hepatositlere farklılaşmayı indüklemek için modifiye edilmiştir. Burada, insan iPS hücrelerinden türetilen hepatositlerin güncel 2D farklılaşması ve saflaştırılması için prosedürlerin ayrıntıları açıklanmaktadır.

<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

İnsan embriyonik kök (ES) ve indüklenmiş pluripotent kök (iPS) hücreleri, sınırsız çoğalmaları ve pluripotent özellikleri nedeniyle ciddi şekilde hastalıklı organları tedavi etmek için hücre bazlı rejeneratif tıpta potansiyel uygulamalara sahiptir. Bununla birlikte, insan ES/iPS hücrelerini %100 saf hedef hücre tiplerine ayırmak, çevreye karşı yüksek hassasiyetleri nedeniyle zordur. Transplantasyondan sonra tümörijenez, kontamine olmuş, çoğalan ve farklılaşmamış hücrelerden kaynaklanır ve bu da rejeneratif tıbbın güvenli bir şekilde gerçekleştirilmesi için yüksek saflaştırma teknolojisini gerekli kılar. Tümör oluşumu riskini azaltmak için, insan iPS hücresinden türetilen hepatositler için yüksek saflaştırma teknolojisi geliştirilmiştir. Yöntem, genetik modifikasyon olmadan yüksek mitokondriyal içerik ve hücre yüzeyi işaretleyicisi ALCAM'ın (aktive edilmiş lökosit hücre adezyon molekülü) bir kombinasyonunu kullanarak FACS (floresanla aktive edilen hücre sıralama) kullanır. Bu yöntem kullanılarak saflaştırılan hepatositlerin %97 ± %0.38'i (n=5) albümin protein ekspresyonu sergilemiştir. Bu makale, insan iPS hücreleri için hepatositlere en güncel iki boyutlu farklılaşma yöntemine uygulandığı gibi, bu yöntem için ayrıntılı prosedürler sağlamayı amaçlamaktadır.

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.

Yazar Spotlight: Rejeneratif Tıp için İnsan Kaynaklı Pluripotent Kök Hücrelerden Hepatosit Saflaştırmasının İlerletilmesi

İnsan Kaynaklı Pluripotent Kök Hücrelerden Türetilen Hepatositleri Saflaştırmak İçin Hücre Sıralamasında Canlı Mitokondri Boyamanın Uygulanması

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.

İnsan iPS hücrelerinin 2D kültür (Şekil 1A) ve temsili hücre özellikleri (Şekil 1B) yoluyla hepatositlere farklılaşmasını indükleyen işlemlerin zaman çizelgesi gösterilmektedir. Yaklaşık olarak farklılaşma 12. günde, hücreler, hepatositlerin karakteristiği olan poligonal hücre şekilleri ve yuvarlak çekirdekler sergilemeye başladı. Bazı hepatositler ayrıca çoklu çekirdeklenme gösterdi.
Farklılaşma 27....

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

Pluripotent kök hücrelerden türetilen hepatositler, mitokondriyal ve aktive edilmiş lökosit hücre adezyon molekülünün (CD166 olarak da bilinen ALCAM) bir kombinasyonu kullanılarak hücre sıralama yoluyla saflaştırılabilir.

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

Araştırmamızın kapsamı, insan kaynaklı pluripotent kök hücre kaynaklı hepatositleri genetik modifikasyon olmadan saflaştırmaktır. İnsan iPS hücrelerinden farklılaşmış olanlar arasında, farklılaşmamış hücrelerle ilişkili tümörjenik risk konusunu ele almayı amaçlıyoruz. Mevcut deneysel zorluklardan biri, sıralamanın mekanik depolanması nedeniyle düşük hücre saflığıdır.Büyük ölçekli saflaştırma için hepatositlerin benzersiz metabolizma özelliklerini kullanan yeni bir saflaştırma yöntemi geliştiriyoruz. Hepatik progenitör hücreler için hücre dışı bir sıralama yöntemi, saf hepatositler elde edemez. Olgun hepatositler için bir diğeri, işaretleyici proteinin sınırlı ekspresyonu nedeniyle hepatositlerin sadece küçük bir kısmını tedavi edebilir.Buna karşılık, yaklaşımımız daha yüksek bir arife ile farklı olgunlaşma seviyelerinde hepatositler elde edebilir. İnsan iPS hücresinden türetilen hepatositler son derece olgunlaşmamıştır ve saflaştırmadan sonra daha azalmış hepatosit makro ekspresyonu gösterir. Gelecekteki hedefimiz, organoidal teknolojiyi ve rejeneratif tıp ve ilaç keşfindeki günlük gelişmeleri kullanarak hepatosit modülasyonunu geliştirmektir.

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.

Biyoloji

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.

İnsan Kaynaklı Pluripotent Kök Hücrelerin 2D Kültürlerde Hepatik Farklılaşması

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.

İnsan Kaynaklı Pluripotent Kök Hücrelerden Elde Edilen Hepatositlerin Saflaştırılması için Hücre Hazırlığı

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.