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

Finansieringen blev leveret af National Institutes of Health (NIH: K01-DK087814-01A1 til D.L.S.), California State University Program for uddannelse og forskning inden for bioteknologi (CSUPERB: Molekylær kontrol af hvirveldyr hæmatopoietisk Niche til D.L.S.) og studier på kontoret på California State University Chico (til A.C.B.).

Institutionelle Animal Care og brug udvalg (IACUC) advisory board på California State University, Chico, godkendt alle nedenfor beskrevne metoder.
1. blegemiddel 48 hpf zebrafisk embryoner
<ol><li>Med 15 cm (w) x 15 cm (l) x 7 cm (h) plastbeholdere oprette 3 wash stationer: 1) med 1 mL 5% blegemiddel i 1000 mL embryo medium (E3, se tabel 1), 2) med steril E3 og 3) med steril E3. Sikre, at hver beholder 500 mL af opløsning til dykke embryoner i. Bemærk: For hver betinge...

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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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Zebrafisk modelsystemet er blevet en effektiv, effektiv og billig model til at studere primitive og endelige hvirveldyr bloddannelsen. Generation af assays, der er hurtig, billig, og præsentere nogle tekniske vanskeligheder kan udnyttes til at teste små molekyler, analysere mutant embryoner og belyse molekylære veje vigtigt for HSPC biologi. In vitro- plating af HSPCs fra voksne zebrafisk er en effektiv metode til at studere mutagenese, cytokiner og hæmatopoietisk defekter. Denne protokol bygger på disse un...

Bloddannelsen er processen med at gøre mange modne blodceller kræves for en organisme overlevelse. Det er en vigtig udviklingsproces, der involverer differentiering af hæmatopoietisk stamceller (HSCs) ind i en række udviklingshæmmede begrænset celletyper, der omfatter modne blod. Disse HSCs skal selv forny, således at systemet aldrig er opbrugt, og de skal fortsætte fra begyndelsen embryonale udvikling indtil døden. I hvirveldyr, konstant differentiering og spredning af hæmatopoietisk stilk og stamfader celler (HSPCs) er nødvendige for at tilstrækkeligt genbestilles fleste af blodlegemer, der er post mitotiske og bliver genanvendt hver dag. HSCs genererer modne blodceller af første differentiering i delmængder af begrænset stamceller; fælles lymfoide stamceller (CLPs)1, som i sidste ende producerer T-, B- og NK celler, og fælles myeloide stamceller (Kystforvaltningsplaner)2 , der genererer granulocytter, erytrocytter, makrofager og megakaryocytes. Disse ophav er forpligtet til at generere specifikke celle slægter, og yderligere differentiere i flere udviklingshæmmede begrænset stamceller som myeloid erythroid ophav (MEP'erne) at genererer erytrocytter og trombocytter eller granulocyt makrofag stamfaderen (GMPs), der genererer basofile, eosinofile, neutrofiler og makrofager2. At identificere og isolere disse stamfaderen giver identifikation af vigtige molekylære veje involveret i hæmatopoietisk differentiering og mange hæmatopoietisk sygdomme såsom leukæmi opstår når disse stamceller ikke korrekt differentiere.
I de seneste par årtier, er zebrafisk (Danio rerio) modelsystemet blevet en vigtig forskningsværktøj til embryonale og voksne hæmatopoietisk undersøgelser. Zebrafisk kan efterproeve genetisk analyse og er Fylogenetisk laveste hvirveldyr model arter, som har en lignende vaskulatur og hæmatopoietisk systemet til mennesker. Zebrafisk embryoner udvikle ex utero, og inden for 48 timer post befrugtning (hpf) generere HSPCs3,4,5,6,7,8. Zebrafisk er også yderst frugtbar, med hunnerne om over 100 æg i en enkelt kobling, giver mulighed for store stikprøvestørrelser og eksperimenterende replikering. Zebrafisk embryoner er optisk gennemsigtige, giver mulighed for mikroskopiske visualisering af hæmatopoietisk systemet. Flere fluorescerende transgene linjer af zebrafisk mærkning HSCs som runx1: EGFP fiske9, cd41: EGFP fiske10, og kdrl:mCherry; cmyb: normal god landbrugspraksis3 dobbelt-positive dyr, giver mulighed for live, real-time visualisering af HSC fremkomsten og ekspansion i vivo3,4,7,8, 9. Zebrafisks hurtig generation tid og udvikling ex utero har ført til dens anvendelse i mutagenese undersøgelser11,12,13,14,15 og stof screening16,17,18,19,20 til forbindelser, der holder terapeutiske lovende for menneskelige blodsygdomme. Bevarelse af hæmatopoietisk systemet, tilstedeværelse og nem udvikling af transgene linjer og hurtig regenerering tid har generelt gjort zebrafisk en billig, hurtig, fleksibel og ideel model for hæmatopoietisk undersøgelser.
Talrige metoder til isolering og test HSCs er blevet udviklet i pattedyr hæmatopoietisk systemer. Efterforskere kan udnytte en kombination af celle overfladen receptorer til at markere HSCs21,22,23,24, samt udnytte evne til HSCs at efflux farvestof25,26. Efter at de er mærket, tillader fluorescens-aktiveret celle sortering (FACS) deres fysiske adskillelse. Beviser, at en celle er en HSC kræver bestråling af en vært dyret at ødelægge endogene HSPCs, omplantning formodede HSCs, og observere langsigtede, multi lineage rekonstituering af alle modne blod celletyper. Disse assays fungerer godt i mus, som der er mange celle-overflade antistoffer mod hæmatopoietisk celler og indavlede mus stammer, der letter immun matchende til transplantation. Par zebrafisk hæmatopoietisk celle-overflade antistoffer har dog været genererede27, hindre identifikation og isolering af HSCs. Den mest almindelige måde at markere og isolere zebrafisk HSCs er med transgene dyr, hvorved en celle-specifikke promotor sekvens er drivkraften bag en fluorescerende proteiner udtryk. Undersøgelser har visualiseret og optalt HSCs i den ventrale mur af dorsal aorta med mikroskopi udnytte denne teknik3,4,8,9. Andre laboratorier har genereret klonede stammer af zebrafisk28,29 og har udført vellykkede transplantationer i MHC-matchede dyr30. Men disse teknikker er omkostningseffektivt uoverkommelige for mange laboratorier, er teknisk vanskeligt, og er tidskrævende. For at løse disse problemer, har laboratorier, der genereret flere in vitro- assays til at teste for forekomst, spredning priser og differentiering kapaciteten af HSPCs31,32,33, 34,35,36. Disse assays vis spredning og differentiering af HSPCs in vitro-31,32,33,34,35,36, redning af hæmatopoietisk defekter36, og en effektiv metode til at opdage og teste cytokiner33,34,35. De har også været udnyttet til at identificere gener ansvarlige for HSPC biologi31,32. I denne undersøgelse tager vi disse assays et skridt videre og giver mulighed for kvantificering af HSPCs i et embryo under udvikling zebrafisk. Disse undersøgelser kan også udnyttes til at kvantificere antallet af HSPCs i mutant dyr og dyr behandlet med hæmatopoietisk-forstyrrende stoffer. I bund og grund disse assays er hurtig, til stede nogle tekniske udfordringer, og billige måder at kvantificere HSPC numre, undersøge deres spredning og undersøge blokke i differentiering.

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

Bloddannelsen er en afgørende cellulære proces hvor hæmatopoietisk stilk og stamfader celler (HSPCs) differentiere i mange forskellige celle slægter, der omfatter modne blod. Isolering og identifikation af disse HSPCs er vanskelig, fordi de er defineret ex post facto; de kan kun defineres efter deres differentiering af specifikke celle lineages. I de seneste par årtier, er zebrafisk (Danio rerio) blevet en model organisme at studere bloddannelsen. Zebrafisk embryoner udvikle ex utero, og af 48 timer efter befrugtning (hpf) har genereret definitive HSPCs. Assays for at vurdere HSPC differentiering og spredning kapaciteter er blevet udviklet, udnytte transplantation og efterfølgende rekonstituering af hæmatopoietisk systemet ud over visualisere specialiserede transgene linjer med Konfokal mikroskopi. Men disse assays er omkostningerne uoverkommelige, teknisk vanskeligt og tidskrævende for mange laboratorier. Udvikling af en in vitro- model til at vurdere HSPCs ville være omkostningseffektive, hurtigere, og færre vanskeligheder i forhold til tidligere beskrevne metoder, så laboratorier til hurtigt at vurdere mutagenese og narkotika skærme, der påvirker HSPC biologi. Denne roman i vitro assay at vurdere HSPCs er udført af plating dissocieres hele zebrafisk embryoner og tilføje eksogene faktorer, der fremmer kun HSPC differentiering og spredning. Embryoner er adskilles i enkelt celler og belagt med HSPC-støttende koloni stimulerende faktorer, der forårsager dem til at generere koloni danner enheder (CFUs) der opstår fra en enkelt stamfader celle. Disse assays skal tillade mere omhyggelig undersøgelse af de molekylære veje ansvarlig for HSPC spredning, differentiering og regulering, som giver forskere til at forstå fundament for hvirveldyr bloddannelsen og dens dysregulering under sygdom.

For at vurdere HSPC numre i embryonale zebrafisk, var 48 hpf embryoner fordøjet, forgyldt i methylcellulose med eksogene hæmatopoietisk-støttende vækstfaktorer og inkuberes i 7 dage (fig. 1A). Efter 7 dage blev koloni danner enheder (CFUs) optalt (figur 1B) og afbildet (figur 1C). Ved at kontrollere de forskellige cytokiner tilføjet, man kan sty...

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 præsenterer vi en enkel metode til at kvantificere hæmatopoietisk stilk og stamfader celler (HSPCs) i embryonale zebrafisk. HSPCs fra dissocierede zebrafisk er belagt i methylcellulose med understøttende faktorer, differentiere til modne blod. Dette giver mulighed for påvisning af blod pixeldefekter og giver mulighed for narkotika screening for at foretages nemt.

Udvikling af et In Vitro Assay at kvantificere hæmatopoietisk stilk og stamfader celler (HSPCs) i at udvikle zebrafisk embryoner

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 Induktion af humane neurale stamceller fra perifert blod hæmatopoietiske 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

Generation af Parabiotic zebrafisk embryoner ved Kirurgisk Fusion af Udvikling 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

Instrueret differentiering af Primitive og endelige hæmatopoietisk ophav fra humane pluripotente 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

Induktion af hypoxi i levende frø og zebrafiskembryoner

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

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Encellede Photoconversion i levende intakt zebrafisk

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 ...

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

Beregningsmæssige analyse af Caenorhabditis elegans kønscelleoverførsel at studere Distribution kerner, proteiner og cytoskelettet

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

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I Vitro Generation af Somite derivater fra menneskelige induceret celler pluripotente stamceller

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

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

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