Name: development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos
Uploaded: 2017-11-30T15:00:00
Description: Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Midler ble gitt av National Institutes of Health (NIH: K01-DK087814-01A1 til D.L.S.), California State University programmet for utdanning og forskning i bioteknologi (CSUPERB: molekylære kontroll av virveldyr blodkreft nisje til D.L.S.) og fra diplomstudium kontoret i California State University Chico (til A.C.B.).

Institusjonelle Animal Care og bruk Committee (IACUC) advisory board på California State University i Chico, godkjent alle metodene som er beskrevet nedenfor.
1. blekemiddel 48 hpf sebrafisk embryo
<ol><li>Med 15 cm (w) x 15 cm (l) x 7 cm (h) plastbeholdere opprette 3 vaskestasjoner: 1) med 1 mL av 5% blekemiddel i 1000 mL embryoet medium (E3, se tabell 1), 2) med sterilt E3, og 3) med sterilt E3. Kontroller at hver beholder har 500 mL løsning å senke embryoene i. Merk...

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	<li>Kondo, M., Weissman, I. L., Akashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+clonogenic+common+lymphoid+progenitors+in+mouse+bone+marrow.">Identification of clonogenic common lymphoid progenitors in mouse bone marrow.</a> Cell. 91, 661-672 (1997).</li><li>Akashi, K., Traver, D., Miyamoto, T., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+clonogenic+common+myeloid+progenitor+that+gives+rise+to+all+myeloid+lineages.">A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.</a> Nature. 404, 193-197 (2000).</li><li>Bertrand, J. 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E., 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=Prostaglandin+E2+regulates+vertebrate+haematopoietic+stem+cell+homeostasis.">Prostaglandin E2 regulates vertebrate haematopoietic stem cell homeostasis.</a> Nature. 447, 1007-1011 (2007).</li><li>Astuti, 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=A+Functional+Bioluminescent+Zebrafish+Screen+for+Enhancing+Hematopoietic+Cell+Homing.">A Functional Bioluminescent Zebrafish Screen for Enhancing Hematopoietic Cell Homing.</a> Stem Cell Reports. 8, 177-190 (2017).</li><li>Kiel, M. J., 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=SLAM+family+receptors+distinguish+hematopoietic+stem+and+progenitor+cells+and+reveal+endothelial+niches+for+stem+cells.">SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells.</a> Cell. 121, 1109-1121 (2005).</li><li>Spangrude, G. J., Heimfeld, S., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+mouse+hematopoietic+stem+cells.">Purification and characterization of mouse hematopoietic stem cells.</a> Science. 241, 58-62 (1988).</li><li>Morrison, S. J., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long-term+repopulating+subset+of+hematopoietic+stem+cells+is+deterministic+and+isolatable+by+phenotype.">The long-term repopulating subset of hematopoietic stem cells is deterministic and isolatable by phenotype.</a> Immunity. 1, 661-673 (1994).</li><li>Osawa, M., Hanada, K., Hamada, H., Nakauchi, H. <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+lymphohematopoietic+reconstitution+by+a+single+CD34-low/negative+hematopoietic+stem+cell.">Long-term lymphohematopoietic reconstitution by a single CD34-low/negative hematopoietic stem cell.</a> Science. 273, 242-245 (1996).</li><li>Goodell, M. 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=Dye+efflux+studies+suggest+that+hematopoietic+stem+cells+expressing+low+or+undetectable+levels+of+CD34+antigen+exist+in+multiple+species.">Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.</a> Nat Med. 3, 1337-1345 (1997).</li><li>Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> J Exp Med. 183, 1797-1806 (1996).</li><li>Gansner, J. 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Sebrafisk modell systemet har blitt en effektiv, effektiv og rimelig modell for å studere primitive og definitive virveldyr hematopoiesis. Generasjon av analyser som er rask, billig, og presentere noen tekniske problemer kan utnyttes for testing små molekyler, analysere mutant embryoer og Klargjørende molekylær trasé viktig for HSPC biologi. In vitro plating av HSPCs fra voksen sebrafisk er en effektiv metode for å studere mutagenese cytokiner og blodkreft defekter. Denne protokollen bygger på disse studi...

Hematopoiesis er prosessen med å lage mange eldre blod celler kreves for en organismes overlevelse. Det er en nøkkel utviklingsprosess som involverer differensiering av blodkreft stamceller (HSCs) til en rekke developmentally begrenset celletyper som utgjør modne blod. Disse HSCs må selv fornye slik at systemet aldri er oppbrukt og de må beholdes fra embryonale utviklingen til død. I virveldyr, konstant differensiering og spredning av blodkreft stilk og stamfar celler (HSPCs) er nødvendig for å tilstrekkelig fylle fleste blod celler som post mitotisk og blir resirkulert hver dag. HSCs genererer eldre blod celler ved første skille i undergrupper av begrenset stamfar celler. vanlige lymfoide progenitors (CLPs)1, som til slutt produsere T, B og NK celler, og vanlige myelogen progenitors (CMPs)2 som genererer granulocytter, erytrocytter, makrofager og megakaryocytes. Disse progenitors er forpliktet til å generere bestemt celle overleveringslinjer og ytterligere skille ut flere developmentally begrenset stamfar celler som myelogen erythroid progenitors (parlamentet) som genererer erytrocytter og blodplater, eller granulocytt macrophage progenitors (GMPs) som genererer basofile, eosinofile, nøytrofile og makrofager2. Identifisere og isolere disse progenitors lar identifikasjon av viktige molekylær veier involvert i blodkreft differensiering og mange blodkreft sykdommer som leukemi oppstår når disse progenitor celler ikke riktig skille.
De siste tiårene, har sebrafisk (Danio rerio) modell systemet blitt en viktig forskningsverktøy for embryonale og voksen blodkreft studier. Sebrafisk er mottakelig for genetisk analyse og phylogenetically laveste virveldyr modell arter som har en lignende blodkar og blodkreft system for mennesker. Sebrafisk embryo utvikle ex utero, og innen 48 timer post generere befruktning (hpf) HSPCs3,4,5,6,7,8. Sebrafisk er også meget fruktbar, kvinner legge over 100 egg i en enkelt clutch, slik at store og eksperimentelle replikering. Sebrafisk embryoer er optisk gjennomsiktige, slik at mikroskopiske visualisering av blodkreft systemet. Flere fluorescerende transgene linjer med sebrafisk merking HSCs som runx1: EGFP fisk9, cd41: EGFP fisk10, og kdrl:mCherry; cmyb: GFP3 dobbel-positive dyr, tillate live, sanntids visualisering av HSC fremveksten og ekspansjon i vivo3,4,7,8, 9. Den sebrafisk rask generasjon tid og utvikling ex utero har ført til bruk i mutagenese studier11,12,13,14,15 og narkotika screening16,17,18,19,20 for forbindelser som holder terapeutiske løftet for menneskelig blod lidelser. Total, bevaring av blodkreft systemet, tilstedeværelse og enkel utvikling av transgene linjer og rask regenerering tid har gjort sebrafisk en billig, rask, fleksibel og ideelle modell for blodkreft studier.
Mange metoder for å isolere og testing HSCs har blitt utviklet i pattedyr blodkreft systemer. Etterforskerne kan bruke en kombinasjon av celleoverflaten reseptorer å merke HSCs21,22,23,24, i tillegg til å utnytte muligheten for HSCs å middelklasseinnbyggere fargestoff25,26. Etter at de er merket, kan aktiveres av fluorescens celle sortering (FACS) deres fysisk separasjon. Krever bevise at en celle er en HSC irradiating en vert dyr for å ødelegge endogene HSPCs, transplanting antatte HSCs, og observere langsiktige, flere avstamning rekonstituering alle eldre typer blodceller. Disse analyser fungere godt i mus, som det er mange celle-overflate antistoffer mot blodkreft cellene og innavlet musen stammer som letter immun matchende for transplantasjon. Men har noen sebrafisk blodkreft celle-overflate antistoffer vært generert27, hindre identifisering og isolering av HSCs. Den vanligste måten å merke og isolere sebrafisk HSCs er med transgene dyr, der en celle-spesifikke promoter sekvens kjører et fluorescerende protein uttrykk. Studier har visualisert og nummerert HSCs i ventrale veggen av dorsalis med mikroskopi utnytte denne teknikken3,4,8,9. Andre laboratorier har generert klonal stammer sebrafisk28,29 og har utført vellykket transplantasjon i MHC-matchet dyr30. Men disse teknikkene er kostnadseffektivt uoverkommelige for mange laboratorier, teknisk vanskelig, og er tidkrevende. For å løse disse problemene, har laboratorier generert flere i vitro analyser til å teste tilstedeværelsen, prisene spredning og differensiering kapasiteten HSPCs31,32,33, 34,35,36. Disse analyser viser spredning og differensiering av HSPCs i vitro31,32,33,34,35,36, redning av blodkreft defekter36og effektiv metode for å oppdage og testing cytokiner33,34,35. De har også blitt benyttet for å identifisere gener ansvarlig for HSPC biologi31,32. I denne studien ta vi disse analyser et skritt videre, slik at kvantifisering av HSPCs i en utvikling sebrafisk fosteret. Disse analyser kan benyttes for å quantitate antall HSPCs i mutant dyr og dyr behandlet med blodkreft-nedbrytende narkotika. I hovedsak disse analyser er rask, presentere noen tekniske utfordringer, og rimelige måter å quantitate HSPC tall, undersøke deres spredning og undersøke blokker i differensiering.

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		<tr height="40">
			<td height="40" style="height:40px;width:341px;">10% Bovine Serum Albumin in Iscove&#39;s MDM</td>
			<td style="width:115px;">StemCell Technologies&#160;</td>
			<td style="width:119px;">9300</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">DPBS (10x) with Calcium2+ and Magnesium2+</td>
			<td style="width:115px;">Life Technologies</td>
			<td style="width:119px;">14080-055</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">HyClone PBS (1x)</td>
			<td style="width:115px;">GE Healthcare Life Sciences</td>
			<td style="width:119px;">sh30256.01</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">&#160;DMEM 500 mL</td>
			<td style="width:115px;">Corning CellGrow</td>
			<td style="width:119px;">10-017-CV</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">&#160;Ham&#39;s F12 500 mL</td>
			<td style="width:115px;">Corning CellGrow</td>
			<td style="width:119px;">10-080-CV</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">FBS 500 mL</td>
			<td style="width:115px;">Gemini Bio-Products</td>
			<td style="width:119px;">100-108</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">HEPES 100 mL (1 M)</td>
			<td style="width:115px;">Gibco life technologies</td>
			<td style="width:119px;">15-630-080</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Penicillin/streptomycin (5000 U/mL 
			and 5000 mg/mL) with L-Glutamine (200 mM)</td>
			<td style="width:115px;">Corning Mediatech</td>
			<td style="width:119px;">30-009-CI</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Gentamycin Sulfate 10 mL (50 mg/mL)</td>
			<td style="width:115px;">Corning Mediatech</td>
			<td style="width:119px;">30-005-CR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:341px;">1.5 mL MCF tube</td>
			<td style="width:115px;">FisherBrand</td>
			<td style="width:119px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">3 mL 23gx1 injection needle with Luer lock</td>
			<td style="width:115px;">BD Safety Glide</td>
			<td style="width:119px;">305905</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">5 mL polystyrene round bottom tube with cell strainer cap</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">352235</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:341px;">Methocel MC</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td style="width:119px;">64630</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">14 mL Polystyrene round bottom tube</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">352057</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">10 mm polystyrene easygrip Petri dish</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">351008</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Librease TM</td>
			<td style="width:115px;">Roche Sigma-Aldrich</td>
			<td>5401119001</td>
			<td>dissociation protease</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">Pronase</td>
			<td style="width:115px;">Roche Sigma-Aldrich</td>
			<td style="width:119px;">11459643001</td>
			<td>dechorionation protease</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:341px;">15 cm Petri dish</td>
			<td style="width:115px;">Corning Falcon</td>
			<td style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:341px;">AB</td>
			<td style="width:115px;">Zebrafish International Resource Center (ZIRC)</td>
			<td style="width:119px;">ZL1</td>
			<td>zebrafish strain used</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:341px;">Dithiolthreitol (DTT)</td>
			<td style="width:115px;">Sigma-Aldrich</td>
			<td>646563</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos

Hematopoiesis er en viktig mobilnettet prosess der blodkreft stilk og stamfar celler (HSPCs) skille ut en rekke ulike celle linjene som utgjør modne blod. Isolasjon og identifikasjon av disse HSPCs er vanskelig fordi de definerte ex post facto; de kan bare defineres etter deres differensiering inn bestemt celle overleveringslinjer. De siste tiårene, har sebrafisk (Danio rerio) blitt en modell organisme til å studere hematopoiesis. Sebrafisk embryo utvikle ex uteroog av 48 timer etter befruktning (hpf) har generert definitive HSPCs. analyser for å vurdere HSPC differensiering og spredning evner er utviklet, utnytte transplantasjon og påfølgende rekonstituering blodkreft systemet i tillegg til visualisere spesialiserte transgene linjer med AC confocal mikroskopi. Men er disse analyser kostnader uoverkommelige, teknisk vanskelig og tidkrevende for mange laboratorier. Utvikling av en i vitro modell å vurdere HSPCs ville være kostnadseffektive, raskere, og presentere færre problemer sammenlignet med tidligere beskrevet metoder, gir laboratorier for å vurdere mutagenese og narkotika skjermer som påvirker HSPC biologi. Denne romanen i vitro analysen å vurdere HSPCs utføres av plating dissosiert hele sebrafisk embryoer og legge ytre faktorer som fremmer bare HSPC differensiering og spredning. Embryo er atskilt i enkeltceller og belagt med HSPC-støttende koloni stimulerende faktorer som forårsaker dem til å generere kolonien danner enheter (CFUs) som oppstår fra en enkelt stamfar celle. Disse analyser bør tillate mer nøye undersøkelse av molekylære veier HSPC spredning, differensiering og regulering, som tillater forskere å forstå grunnlaget for virveldyrenes hematopoiesis og dens feilregulering under sykdom.

For å vurdere HSPC tall i embryonale sebrafisk, var 48 hpf embryo fordøyd, belagt i methylcellulose med ytre blodkreft-støttende vekstfaktorer og ruges i 7 dager (figur 1A). Etter 7 dager var koloni danner enheter (CFUs) opplistet (figur 1B) og fotografert (figur 1C). Ved å kontrollere de ulike cytokiner lagt, man kan kontrollere hvilke typer kol...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Her presenterer vi en enkel metode for å quantitate blodkreft stilk og stamfar celler (HSPCs) i embryonale sebrafisk. HSPCs fra avstand sebrafisk er belagt i methylcellulose med støttende faktorer, skille i eldre blod. Dette gjør påvisning av blod defekter og lar narkotikarelaterte screening for å utføres enkelt.

Utvikling av In Vitro analysen å Quantitate blodkreft stilk og stamfar celler (HSPCs) i utvikle sebrafisk embryo

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Direkte Induction of Human Neural stamceller fra perifert blod Hematopoietiske stamceller

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Generering av Parabiotic Sebrafisk embryoer ved Kirurgisk Fusion of Utvikling Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

Rettet differensiering av Primitive og Definitive blodkreft Progenitors fra menneskelige Pluripotent stamceller

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Induksjon av hypoksi i levende frosk og sebrafiskembryoer

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

Kvantitativ hele-mount Immunofluorescence analyse av Cardiac stamfar populasjoner i Mouse embryoer

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Single-cell Photoconversion in Living Intact Zebrafish

Encellede Photoconversion i levende intakt sebrafisk

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

Beregningsorientert analyse av Caenorhabditis elegans Germline å studere distribusjon av kjerner, proteiner og Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

I Vitro generasjon av Somite derivater fra menneske indusert stamceller Pluripotent

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

Vurdering av oksidativ skade i primær mus okulære overflateceller / stamceller som svar på ultrafiolett-C (UV-C) skade

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...