Name: Autor destacado: Avance de la purificación de hepatocitos a partir de células madre pluripotentes inducidas humanas para la medicina regenerativa
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

Este trabajo contó con el apoyo de la subvención número [23390072] del Ministerio de Educación, Cultura, Deporte, Ciencia y Tecnología.

En este estudio se utilizaron células iPS humanas obtenidas comercialmente (cepa 253G1) (véase la Tabla de Materiales).
1. Mantenimiento de las células iPS humanas
<ol><li>Mantener las células iPS humanas en condiciones libres de alimentador en medio AK02 en placas de cultivo recubiertas con iMatrix-511 de 0,25 μg/cm2 (ver Tabla de Materiales).</li>
</ol>2. Diferenciación hepática de células i...

Purificación de hepatocitos derivados de iPSC humanos mediante clasificación mitocondrial-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. 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>

Debido a sus funciones en el metabolismo de nutrientes y la desintoxicación, los hepatocitos poseen un número relativamente grande de mitocondrias en comparación con otros tipos de células15. ALCAM es un miembro de la superfamilia de las inmunoglobulinas y desempeña un papel en la adhesión y migración celular. Se expresa en varios tipos de células, incluyendo células hepáticas, epiteliales, linfocíticas, mieloides, fibroblastos y neuronales16. Mediante la utiliza...

Las células madre pluripotentes embrionarias e inducidas (ES e iPS, respectivamente) se consideran fuentes celulares prometedoras para terapias regenerativas. Sin embargo, la eficiencia de diferenciar estas células en tipos específicos de células objetivo puede variar, incluso cuando se utiliza la misma línea celular, protocolo y experimentador 1,2,3,4. Esta variabilidad puede atribuirse a la alta sensibilidad de las células ES/iPS humanas a su entorno. Por lo tanto, actualmente es difícil obtener de manera consistente células diana puras. Para lograr una medicina regenerativa altamente segura, es crucial eliminar las células proliferativas y las células madre indiferenciadas en las células terapéuticas, y la tecnología avanzada de purificación para las células diana es esencial 5,6,7.
Un clasificador de células es un dispositivo que analiza instantáneamente las células individuales y clasifica las células vivas de interés en función de la intensidad de la señal fluorescente, lo que ofrece una solución prometedora. Esto se puede lograr mediante la tinción de anticuerpos de marcadores de superficie específicos del tipo celular o mediante la utilización de expresiones de genes reporteros específicos del tipo celular. Utilizando esta técnica, existen varios reportes sobre métodos para purificar cardiomiocitosderivados de células madre pluripotentes 8,9,10 y hepatocitos 11,12. Hattori et al. desarrollaron un innovador método de purificación mitocondrial utilizando un clasificador de células13. Aprovechando el hecho de que los cardiomiocitos tienen altas demandas de energía a través de la actividad mitocondrial, la tinción de las células con el colorante indicativo de mitocondrias vivas TMRM (éster metílico de tetrametilrodamina) se puede utilizar para marcar y purificar altamente cardiomiocitos por FACS a partir de cuerpos embrioides derivados de células madre embrionarias humanas que contienen varios tipos de células. La ausencia de tumorigénesis se confirmó mediante ensayos de formación de teratomas con los cardiomiocitos purificados. Además, Yamashita et al. descubrieron inesperadamente un método para purificar hepatocitos de cuerpos embrioides derivados de células madre embrionarias humanas mediante el aislamiento de fracciones con alta actividad mitocondrial y expresión positiva de ALCAM14. La justificación de este método es que los hepatocitos también tienen un número relativamente alto de mitocondrias debido a su alto consumo de ATP para el metabolismo de nutrientes y la desintoxicación15, y los hepatocitos expresan ALCAM, un miembro de la superfamilia de las inmunoglobulinas, que desempeña un papel en la adhesión y migración celular16.
Los métodos previos de alta purificación para hepatocitos derivados de células madre pluripotentes requerían modificaciones genéticas, y los métodos de purificación no genética tenían baja eficiencia17. El método mitocondrial no genético tiene mérito para lograr una alta pureza. Cuando se necesitan células progenitoras hepáticas, se pueden elegir los métodos basados en CD133 y CD13 o Dlk118,19. Aunque la precisión de la tecnología de edición del genoma ha avanzado, el riesgo potencial de cambios genómicos imprevistos (por ejemplo, carcinogénesis) no se puede eliminar por completo. Los métodos basados en la actividad mitocondrial, sin implicar modificación genética, pueden estar libres de tales riesgos.
Como resultado del examen de varios indicadores mitocondriales en cardiomiocitos de ratas neonatales, se observó que la TMRM desapareció completamente en 24 h, mientras que otros colorantes permanecieron durante al menos 5 días13. Además, es importante señalar que TMRM y JC-1 no afectaron la viabilidad celular cuando se evaluaron con el ensayo de bromuro de 3-(4,5-dimetil-tiazol-2-il)-2,5-difeniltetrazolio (MTT), mientras que otros colorantes demostraron efectos variables sobre la viabilidad celular13. TMRM demuestra una mayor seguridad.
Hasta la fecha, se han desarrollado varios métodos de diferenciación bidimensional (2D) como enfoques más eficientes para inducir la diferenciación en comparación con la formación de cuerpos embrioides tridimensionales (3D). Esto se debe a que los compuestos o citocinas inductoras de diferenciación escalonada se pueden administrar uniformemente a las células en un plano 2D, en lugar de en un espacio 3D. En este estudio, el método20 previamente reportado fue modificado para inducir la diferenciación en hepatocitos derivados de células iPS humanas. Aquí, se describen los detalles de los procedimientos para la diferenciación y purificación 2D actualizada de hepatocitos derivados de células iPS humanas.

<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

Las células madre embrionarias humanas (ES) y las células madre pluripotentes inducidas (iPS) tienen aplicaciones potenciales en la medicina regenerativa basada en células para el tratamiento de órganos gravemente enfermos debido a su proliferación ilimitada y propiedades pluripotentes. Sin embargo, diferenciar las células ES/iPS humanas en tipos de células diana 100% puras es un reto debido a su alta sensibilidad al medio ambiente. La tumorigénesis después del trasplante es causada por células contaminadas, proliferantes e indiferenciadas, lo que hace que la tecnología de alta purificación sea esencial para la realización segura de la medicina regenerativa. Para mitigar el riesgo de tumorigénesis, se ha desarrollado una tecnología de alta purificación para hepatocitos derivados de células iPS humanas. El método emplea FACS (clasificación celular activada por fluorescencia) utilizando una combinación de alto contenido mitocondrial y el marcador de superficie celular ALCAM (molécula de adhesión celular leucocitaria activada) sin modificación genética. El 97% ± 0,38% (n = 5) de los hepatocitos purificados mediante este método mostraron expresión de la proteína albúmina. Este artículo tiene como objetivo proporcionar procedimientos detallados para este método, aplicados al método de diferenciación bidimensional más actual para células iPS humanas en hepatocitos.

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.

Autor destacado: Avance de la purificación de hepatocitos a partir de células madre pluripotentes inducidas humanas para la medicina regenerativa

Aplicación de la tinción de mitocondrias vivas en la clasificación celular para purificar hepatocitos derivados de células madre pluripotentes inducidas humanas

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.

Se muestra la cronología de los procesos que inducen a las células iPS humanas a diferenciarse en hepatocitos a través de cultivo 2D (Figura 1A) y las características celulares representativas (Figura 1B). Aproximadamente en el día 12 de diferenciación, las células comenzaron a exhibir formas celulares poligonales y núcleos redondos, característicos de los hepatocitos. Algunos hepatocitos también mostraron multinucleación.
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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

Los hepatocitos derivados de células madre pluripotentes se pueden purificar a través de la clasificación celular, utilizando una combinación de tinción de moléculas de adhesión de células leucocitarias mitocondriales y activadas (ALCAM, también conocida como CD166).

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

El alcance de nuestra investigación es purificar hepatocitos derivados de células madre pluripotentes inducidas humanas, utilizando sin modificación genética. Nuestro objetivo es abordar la cuestión del riesgo tumorigénico asociado a las células indiferenciadas, entre las diferenciadas de las células iPS humanas. Uno de los retos experimentales actuales es la baja pureza de las células, debido al almacenamiento mecánico de la clasificación.Estamos desarrollando un nuevo método de purificación, que utiliza las características metabólicas únicas de los hepatocitos, para la purificación a gran escala. Un método de clasificación no celular para las células progenitoras hepáticas no puede obtener hepatocitos puros. Otro para hepatocitos maduros solo puede tratar una pequeña fracción de hepatocitos, debido a la expresión limitada de la proteína marcada.Por el contrario, nuestro enfoque puede obtener hepatocitos en diferentes niveles de maduración con una mayor eva. Los hepatocitos derivados de células iPS humanas son extremadamente inmaduros y muestran una expresión más reducida de macrocitos después de la purificación. Nuestro objetivo futuro es mejorar la modulación de los hepatocitos utilizando tecnología organoidal y los avances diarios hacia la medicina regenerativa y el descubrimiento de fármacos.

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.

Investigación

Biología

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.

Diferenciación hepática de células madre pluripotentes inducidas por humanos en cultivos 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.

Preparación celular para la purificación de hepatocitos derivados de células madre pluripotentes inducidas por humanos

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.