Name: immunophenotyping and cell sorting of human mks from human primary sources or differentiated in vitro from hematopoietic progenitors
Uploaded: 2021-08-07T15:00:00
Description: Watch this Scientific Journal Video about Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors at JoVE.com

Vi takker Marcos Pérez Basterrechea, Lorena Rodríguez Lorenzo og Begoña García Méndez (HUCA) og Paloma Cerezo, Almudena Payero og María de la Poveda-Colomo (HCSC) for teknisk support. Dette arbejde blev delvist støttet af Medical Grants (Roche SP200221001) til A.B., et RYC-stipendium (RYC-2013-12587; Ministerio de Economía y Competitividad, Spanien) og et I+D 2017-tilskud (SAF2017-85489-P; Ministerio de Ciencia, Innovación y Universidades, Spanien og Fondos FEDER) til L.G., et Severo Ochoa-tilskud (PA-20-PF-BP19-014; Consejería de Ciencia, Innovación y Universidades del Principado de Asturias, Spanien) til P.M.-B. og et intramuralt postdoc-tilskud 2018 (Fundación para la Investigación y la Innovación Biosanitaria de Asturias - FINBA, Oviedo, Spanien) til A.A.-H. Vi takker Reinier van der Linden for at dele sin viden (og tid), især hans kloge råd om multi-farve mærkede-antistof panel mix og enkelt-farve perle kontrol forberedelse.

Der blev udtaget og behandlet fuldblods- og knoglemarvsprøver i overensstemmelse med Helsingfors-erklæringen fra 1964. Fuldblodsprøver blev udtaget fra raske donorer efter at have givet informeret samtykke (ISPA) i en undersøgelse godkendt af vores institutionelle medicinske etiske udvalg (Hospital Universitario Central de Asturias -HUCA-). Knoglemarvsprøver blev fremstillet af knoglemarvssugudsmidsmateriale fra patienter, der blev forvaltet på Dept. of Hematology på Hospitalet Clínico San Carlos (HCSC).
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<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Incredible+Journey:+From+Megakaryocyte+Development+To+Platelet+Formation.">The Incredible Journey: From Megakaryocyte Development To Platelet Formation.</a> Journal Of Cell Biology. 201 (6), 785-796 (2013).</li><li>Eckly, A., 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=Biogenesis+Of+The+Demarcation+Membrane+System+(DMS)+In+Megakaryocytes.">Biogenesis Of The Demarcation Membrane System (DMS) In Megakaryocytes.</a> Blood. 123 (6), 921-930 (2014).</li><li>Couldwell, G., Machlus, K. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Platelet+Biogenesis+In+The+Lung+Circulation.">Platelet Biogenesis In The Lung Circulation.</a> Physiology (Bethesda). 34 (6), 392-401 (2019).</li><li>Nieswandt, B., Stritt, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Megakaryocyte+Rupture+For+Acute+Platelet+Needs.">Megakaryocyte Rupture For Acute Platelet Needs.</a> Journal Of Cell Biology. 209 (3), 327-328 (2015).</li><li>Nishimura, 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=IL-1alpha+Induces+Thrombopoiesis+Through+Megakaryocyte+Rupture+In+Response+To+Acute+Platelet+Needs.">IL-1alpha Induces Thrombopoiesis Through Megakaryocyte Rupture In Response To Acute Platelet Needs.</a> Journal Of Cell Biology. 209 (3), 453-466 (2015).</li><li>Sanjuan-Pla, A., 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=Platelet-Biased+Stem+Cells+Reside+At+The+Apex+Of+The+Haematopoietic+Stem-Cell+Hierarchy.">Platelet-Biased Stem Cells Reside At The Apex Of The Haematopoietic Stem-Cell Hierarchy.</a> Nature. 502 (7470), 232-236 (2013).</li><li>Notta, 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=Distinct+Routes+Of+Lineage+Development+Reshape+The+Human+Blood+Hierarchy+Across+Ontogeny.">Distinct Routes Of Lineage Development Reshape The Human Blood Hierarchy Across Ontogeny.</a> Science. 351 (6269), 2116 (2016).</li><li>Yamamoto, R., 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=Clonal+Analysis+Unveils+Self-Renewing+Lineage-Restricted+Progenitors+Generated+Directly+From+Hematopoietic+Stem+Cells.">Clonal Analysis Unveils Self-Renewing Lineage-Restricted Progenitors Generated Directly From Hematopoietic Stem Cells.</a> Cell. 154 (5), 1112-1126 (2013).</li><li>Wang, 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=Decoding+Human+Megakaryocyte+Development.">Decoding Human Megakaryocyte Development.</a> Cell Stem Cell. , (2020).</li><li>Meinders, M., 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=Repercussion+Of+Megakaryocyte-Specific+Gata1+Loss+On+Megakaryopoiesis+And+The+Hematopoietic+Precursor+Compartment.">Repercussion Of Megakaryocyte-Specific Gata1 Loss On Megakaryopoiesis And The Hematopoietic Precursor Compartment.</a> Plos One. 11 (5), 0154342 (2016).</li><li>Meinders, M., 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=Sp1/Sp3+Transcription+Factors+Regulate+Hallmarks+Of+Megakaryocyte+Maturation+And+Platelet+Formation+And+Function.">Sp1/Sp3 Transcription Factors Regulate Hallmarks Of Megakaryocyte Maturation And Platelet Formation And Function.</a> Blood. 125 (12), 1957-1967 (2015).</li><li>Salunkhe, V. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Prospective+Isolation+Of+Viable,+High+Ploidy+Megakaryocytes+From+Adult+Murine+Bone+Marrow+By+Fluorescence+Activated+Cell+Sorting.">The Prospective Isolation Of Viable, High Ploidy Megakaryocytes From Adult Murine Bone Marrow By Fluorescence Activated Cell Sorting.</a> Methods In Molecular Biology. 1035, 121-133 (2013).</li><li>Tomer, A., Harker, L. A., Burstein, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+Of+Human+Megakaryocytes+By+Fluorescence-Activated+Cell+Sorting.">Purification Of Human Megakaryocytes By Fluorescence-Activated Cell Sorting.</a> Blood. 70 (6), 1735-1742 (1987).</li><li>Martinez-Botia, P., Acebes-Huerta, A., Seghatchian, J., Gutierrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+The+Quest+For+In+Vitro+Platelet+Production+By+Re-Tailoring+The+Concepts+Of+Megakaryocyte+Differentiation.">On The Quest For In Vitro Platelet Production By Re-Tailoring The Concepts Of Megakaryocyte Differentiation.</a> Medicina. 56 (12), (2020).</li><li>Martinez-Botia, P., Acebes-Huerta, A., Seghatchian, J., Gutierrez, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Platelet+Production+For+Transfusion+Purposes:+Where+Are+We+Now.">In Vitro Platelet Production For Transfusion Purposes: Where Are We Now.</a> Transfusion And Apheresis Science. 59 (4), 102864 (2020).</li><li>Butov, K. 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Det meste af forskningen med fokus på studiet af megakaryopoiesis ved flowcytometri er til dato begrænset til identifikation af MK-delsæt, der kun bruger afstamningsspecifikke overflademarkører (dvs.CD42A/CD42B, CD41/CD61), mens tidligere MK-differentieringsstadier er blevet dårligt undersøgt. I denne artikel viser vi en immunophenotyping strategi for at løse en omfattende flow cytometri karakterisering af menneskelige megakaryopoiesis. Samlet set vil vi gerne fremhæve nytten af at kombinere MK specifikk...

Megakaryocytter (MKs) udvikler sig fra hæmatopoietiske stamceller (HSC'er) efter en kompleks proces kaldet megakaryopoiesis, som hovedsageligt orkestreres af hormonet thrombopoietin (TPO). Den klassiske opfattelse af megakaryopoiesis beskriver den cellulære rejse fra HSC'er gennem en række hierarkiske stadier af engagerede forfædre og prækursorceller, hvilket i sidste ende fører til en moden MK. Under modning oplever MKs flere runder af endomitose, udvikler et indviklet intracellulært afgrænsningsmembransystem (DMS), som giver nok membranoverflade til pladeproduktion og effektivt producerer og pakker overfloden af faktorer, der er indeholdt i de forskellige granulater arvet af modne blodplader1,2,3. Som følge heraf er modne MKs store celler (40-60 μm) karakteriseret ved en meget polyploid kerne (når selv >64N). Nylige undersøgelser tyder på alternative ruter,hvorved HSC'er differentierer sig til MKs uden om traditionelle kontrolpunkter for trinvise forpligtelser som reaktion på visse fysiopatologiske tilstande4,5,6,7,8,9,10,11. Disse fund fremhæver, at hæmatopoietisk differentiering mod den modne MK er en kontinuum og adaptiv proces, der reagerer på biologiske behov.
Med den stigende viden om cellebiologien og de molekylære aspekter, der karakteriserer megakaryopoiesis12, er det meste af forskningen dedikeret til studiet af processen ved flowcytometry begrænset til identifikation af modne MKs ved hjælp af afstamningsspecifikke overflademarkører (dvs. CD42A / B, CD41/CD61), mens tidligere MK-differentieringsstadier forbliver uudforskede. Vi har tidligere dokumenteret en strategi for at iscenesætte megakaryopoiesis i mus knoglemarv og knoglemarv-afledte MK kulturer13,14, som vi har tilpasset og anvendt til mennesker15. I denne artikel viser vi en immunophenotyping strategi, der gør det muligt karakterisering af megakaryopoiesis, fra HSC'er til modne MKs, i menneskets primære kilder (knoglemarv -BM- og perifert blod -PB-) eller in vitro kulturer ved hjælp af et panel, der integrerer MK specifikke og ikke-specifikke overflademarkører (CD61, CD42B, CD49B, CD31, KIT og CD71, blandt andre). På trods af sin store størrelse og skrøbelighed kan MKs immunohenotyped ved hjælp af ovennævnte celleoverflademarkører og beriget med fluorescensaktiveret cellesortering under særlige forhold med tryk og dysedymeter for at minimere cellebrud og / eller skade. Denne teknik letter multi-Omics tilgange, med det formål bedre at forstå kompleksiteten af megakaryopoiesis og blodplader produktion i menneskers sundhed og sygdom. Bemærkelsesværdigt, vil det udgøre som et nyttigt redskab til at støtte diagnose og prognose i en klinisk sammenhæng med stigende efterspørgsel.
I dette manuskript dokumenterer vi en strategi for at iscenesætte menneskelige megakaryopoiesis med et panel, der integrerer MK-specifikke og ikke-specifikke overflademarkører fra primære kilder eller genereres in vitro. Derudover leverer vi en protokol til sortering med en fluorescensaktiveret cellesortering, de foretrukne fraktioner og modne MKs (Figur 1). Dette trin er ikke populært, da det er teknisk vanskeligt på grund af MKs's store størrelse og skrøbelighed. Det har dog været ansat både i muse- og human knoglemarvsprøver tidligere og på grund af teknologiske fremskridt med et bedre resultat hver gang16,17,18. Menneskelige primære kilder, hvor MKs eller MK prækursorer kan studeres omfatter knoglemarv, navlestrengsblod og perifert blod, blandt andre. Den korrekte prøvebehandling til isolering af den relevante cellefraktion til analyse af hver prøve er vigtig. Standardprocedurer indarbejdes med nogle overvejelser, der skal tages i betragtning, når man sigter mod undersøgelsen af megakaryopoiesis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>130 micron Nozzle</td>
			<td>BD</td>
			<td>643943</td>
			<td>required for MK sorting</td>
		</tr>
		<tr>
			<td>5810R Centrifuge</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Cell isolation and washes</td>
		</tr>
		<tr>
			<td>A-4-62 Swing Bucket Rotor</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Cell isolation and washes</td>
		</tr>
		<tr>
			<td>Aerospray Pro Hematology Slide Stainer / Cytocentrifuge</td>
			<td>ELITech Group</td>
			<td></td>
			<td>Automatized cytology devise, where slides are stained with Mat-Gr&#252;nwald Giemsa</td>
		</tr>
		<tr>
			<td>CO2 Incubator Galaxy 170 S</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Cell Incubation</td>
		</tr>
		<tr>
			<td>Cytospin 4 Cytocentrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>To prepare cytospins</td>
		</tr>
		<tr>
			<td>FACSAria IIu sorter</td>
			<td>BD</td>
			<td></td>
			<td>Lasers 488-nm and 633-nm</td>
		</tr>
		<tr>
			<td>FACSCanto II flow cytometer</td>
			<td>BD</td>
			<td></td>
			<td>Lasers 488-nm , 633-nm and 405-nm</td>
		</tr>
		<tr>
			<td>Olympus Microscope BX 41</td>
			<td>Olympus</td>
			<td></td>
			<td>Microphotographs</td>
		</tr>
		<tr>
			<td>Olympus Microscope BX 61</td>
			<td>Olympus</td>
			<td></td>
			<td>Microphotographs</td>
		</tr>
		<tr>
			<td>Zoe Fluorescent Cell Imager</td>
			<td>BioRad</td>
			<td></td>
			<td>Microphotographs</td>
		</tr>
		<tr>
			<td>To obtain PBMCs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Lipids Cholesterol Rich from adult bovine serum</td>
			<td>Sigma-Aldrich</td>
			<td>L4646</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Lymphoprep</td>
			<td>Stem Cell Technologies</td>
			<td>#07801</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Recombinant human Erythropoietin (EPO)</td>
			<td>&#160;R&#38;D Systems</td>
			<td>287-TC-500</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Recombinant human stem cell factor (SCF)</td>
			<td>Thermo Fisher Scientific, Gibco&#8482;</td>
			<td>PHC2115</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Recombinant human thrombopoietin (TPO)</td>
			<td>Thermo Fisher Scientific, Gibco&#8482;</td>
			<td>PHC9514</td>
			<td>or TPO receptor agonists</td>
		</tr>
		<tr>
			<td>StemSpan SFEM</td>
			<td>Stem Cell Technologies</td>
			<td>#09650</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow Cytometry Analyses</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Merck</td>
			<td>A7906-100G</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>BD CompBead Anti-Mouse Ig, &#954;/Negative Control Compensation Particles Set</td>
			<td>BD</td>
			<td>552843</td>
			<td>Antibodies for human cells are generally from mouse.</td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm</td>
			<td>BD</td>
			<td>554714</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>BD FACS Accudrop Beads</td>
			<td>BD</td>
			<td>345249</td>
			<td></td>
		</tr>
		<tr>
			<td>CD31 AF-647</td>
			<td>BD</td>
			<td>561654</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD31 FITC</td>
			<td>Immunostep</td>
			<td>31F-100T</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34 FITC</td>
			<td>BD</td>
			<td>555821</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD41 PE</td>
			<td>BD</td>
			<td>555467</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD41 PerCP-Cy5.5</td>
			<td>BD</td>
			<td>333148</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD42A APC</td>
			<td>Immunostep</td>
			<td>42AA-100T</td>
			<td>We observed unspecific binding... that needs to be assessed</td>
		</tr>
		<tr>
			<td>CD42A PE</td>
			<td>BD</td>
			<td>558819</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD42B PerCP</td>
			<td>Biolegend</td>
			<td>303910</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD49B PE</td>
			<td>BD</td>
			<td>555669</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD61 FITC</td>
			<td>BD</td>
			<td>555753</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>CD71 APC-Cy7</td>
			<td>Biolegend</td>
			<td>334109</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Human BD Fc Block</td>
			<td>BD</td>
			<td>564219</td>
			<td>Fc blocking - control</td>
		</tr>
		<tr>
			<td>KIT PE-Cy7</td>
			<td>Biolegend</td>
			<td>313212</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>Lineage Cocktail 2 FITC</td>
			<td>BD</td>
			<td>643397</td>
			<td>Mouse anti-human</td>
		</tr>
		<tr>
			<td>RNAse</td>
			<td>Merck</td>
			<td>R6513</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Triton X-500</td>
			<td>Merck</td>
			<td>93443-500ML</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Cell strainers for sorting</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CellTrics Filters 100 micrometers</td>
			<td>Sysmex</td>
			<td>04-004-2328</td>
			<td>Cell strainers</td>
		</tr>
		<tr>
			<td colspan="4">Note: we do not specify general reagents/chemicals (PBS, EDTA, etc) or disposables (tubes, etc), or reagents specified in previous published and standard protocols&#160; - unless otherwise specified.</td>
		</tr>
	</tbody>
</table>

immunophenotyping and cell sorting of human mks from human primary sources or differentiated in vitro from hematopoietic progenitors

Megakaryocyte (MK) differentiering omfatter en række endomitotiske cyklusser, der resulterer i en meget polyploid (nå selv >64N) og ekstremt store celle (40-60 μm). I modsætning til den hurtigt stigende viden i megakaryopoiesis på cellebiologi og molekylært niveau er karakteriseringen af megakaryopoiesis ved flowcytometry begrænset til identifikation af modne MKs ved hjælp af afstamningsspecifikke overflademarkører, mens tidligere MK-differentieringsstadier forbliver uudforskede. Her præsenterer vi en immunophenotyping strategi, der gør det muligt at identificere successive MK differentieringsstadier, med stigende ploidy status, i menneskets primære kilder eller in vitro kulturer med et panel, der integrerer MK specifikke og ikke-specifikke overflade markører. På trods af sin størrelse og skrøbelighed kan MKs immunophenotyped ved hjælp af ovennævnte panel og beriget med fluorescensaktiveret cellesortering under særlige betingelser for tryk og dysedymeter. Denne tilgang letter multi-Omics undersøgelser med det formål bedre at forstå kompleksiteten af megakaryopoiesis og blodpladeproduktion hos mennesker. En bedre karakterisering af megakaryopoiesis kan udgøre grundlæggende i diagnosen eller prognosen for afstamning-relaterede patologier og malignitet.

Knoglemarv og ploidy I figur 4viservi en repræsentativ immunophenotypinganalyse af megakaryopoiesis i BM-prøver (aspiration) fra patienter. Når vi planlægger den cellulære fraktion mod CD71 og CD31, har vi gated seks hovedpopulationer: CD31- CD71- (rød), CD31- CD71+ (blå), CD31+ CD71- (orange), CD31+ CD71midt (lysegrøn), CD31+ CD71+ (mørk...

Watch this Scientific Journal Video about Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors at JoVE.com

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Her præsenterer vi en immunophenotyping strategi for karakterisering af megakaryocyt differentiering, og viser, hvordan denne strategi tillader sortering af megakaryocytter på forskellige stadier med en fluorescens-aktiveret cellesortering. Metoden kan anvendes på humane primære væv, men også til megakaryocytter genereret i kultur in vitro.

Immunophenotyping og cellesortering af humane MKs fra humane primære kilder eller differentieret in vitro fra hæmatopoietiske forfædre

Megakaryocyte (MK) differentiation encompasses a number of endomitotic cycles that result in a highly polyploid (reaching even&#160;&#62;64N) and ...

Hello.I er Andrea Acebes Huerta.Postdoctoral Forsker ved Platelet Research Lab del af Health Research Institute of fyrstendømmet Asturien, ISPA. Som er beliggende i Oviedo, Spain.Hello.I er Laura Guti

Research

JoVE Journal

Biology

Immunophenotyping and Cell Sorting of Human MKs from Human Primary Sources or Differentiated In Vitro from Hematopoietic Progenitors

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

Effektiv Udledning af menneskelige Hjerte prækursorer og cardiomyocytter fra Pluripotente humane embryonale stamceller med småmolekyle Induktion

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

Fænotypisk analyse og isolering af murine hæmatopoietiske stamceller og Lineage-progenitorer

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

NaÃƒÆ’Ã‚Â¯ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Naive voksne stamceller Isolering fra primære humane fibroblastkulturer

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

Isolering af Primære myofibroblasts fra mus og Human tyktarmsvæv

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Isolering af blod-fartøj-afledt Multipotente Forstadier fra human skeletmuskel

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

Effektiv Generation Menneskelige induceret pluripotente stamceller fra humane somatiske celler med Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...