Name: combining intravital fluorescent microscopy (ivfm) with genetic models to study engraftment dynamics of hematopoietic cells to bone marrow niches
Uploaded: 2017-03-21T13:00:00
Description: Watch this Scientific Journal Video about Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches at JoVE.com

Imaging blev udført i Indiana Center for biologisk mikroskopi på Indiana University, instrueret af Dr. Ken Dunn. Stereotaxisk enheden er en prototype designet og fremstillet af Mark Soonpaa, brønde Center for pædiatrisk forskning. Dette arbejde blev støttet af NIH/R01DK097837-09 (NC), NIH/R01HL068256-05 (NC), NIH/NIDDK1U54DK106846-01 (NC), MPN research Foundation (NC) og CTSI Collaborative projekt IUSM/Notre Dame (NC).

Alle procedurer, der involverer brugen af dyr blev udført med tilladelse af Animal Care og bruge Udvalget af Indiana University School of Medicine. Sikre, at du overholder lovgivningen om dyreforsøg i det land, hvor arbejdet udføres.
1. forberedelse af Mx1CreRBPJ- / - Recipient mus
<ol><li>Cross Mx1-Cre+ mus med RBPJlox/lox mus10 at opnå Mx1-Crepositive RBPJlox/lox mus12 og M...

<ol>
	<li>Carlesso, N., Cardoso, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+regulatory+niches+and+their+role+in+normal+and+malignant+hematopoiesis.">Stem cell regulatory niches and their role in normal and malignant hematopoiesis.</a> Curr Opin Hematol. 17 (4), 281-286 (2010).</li><li>Lo Celso, C., Scadden, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+haematopoietic+stem+cell+niche+at+a+glance.">The haematopoietic stem cell niche at a glance.</a> J Cell Sci. 124 (PT 21), 3529-3535 (2011).</li><li>Calvi, L. 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=Osteoblastic+cells+regulate+the+haematopoietic+stem+cell+niche.">Osteoblastic cells regulate the haematopoietic stem cell niche.</a> Nature. 425 (6960), 841-846 (2003).</li><li>Kiel, M. J., Yilmaz, O. H., Iwashita, T., Terhorst, C., Morrison, S. J. <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 (7), 1109-1121 (2005).</li><li>Zhang, 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=Identification+of+the+haematopoietic+stem+cell+niche+and+control+of+the+niche+size.">Identification of the haematopoietic stem cell niche and control of the niche size.</a> Nature. 425 (6960), 836-841 (2003).</li><li>Mendez-Ferrer, 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=Mesenchymal+and+haematopoietic+stem+cells+form+a+unique+bone+marrow+niche.">Mesenchymal and haematopoietic stem cells form a unique bone marrow niche.</a> Nature. 466 (7308), 829-834 (2010).</li><li>Naveiras, O., 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=Bone-marrow+adipocytes+as+negative+regulators+of+the+haematopoietic+microenvironment.">Bone-marrow adipocytes as negative regulators of the haematopoietic microenvironment.</a> Nature. 460 (7252), 259-263 (2009).</li><li>Scheiermann, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenergic+nerves+govern+circadian+leukocyte+recruitment+to+tissues.">Adrenergic nerves govern circadian leukocyte recruitment to tissues.</a> Immunity. 37 (2), 290-301 (2012).</li><li>Faust, N., Varas, F., Kelly, L. M., Heck, S., Graf, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insertion+of+enhanced+green+fluorescent+protein+into+the+lysozyme+gene+creates+mice+with+fluorescent+granulocytes+and+macrophages.">Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with fluorescent granulocytes and macrophages.</a> Blood. 96 (2), 716-726 (2000).</li><li>Han, 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=Inducible+gene+knockout+of+transcription+factor+recombination+signal+binding+protein-J+reveals+its+essential+role+in+T+versus+B+lineage+decision.">Inducible gene knockout of transcription factor recombination signal binding protein-J reveals its essential role in T versus B lineage decision.</a> Int Immunol. 14 (6), 637-645 (2002).</li><li>Lo Celso, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-animal+tracking+of+individual+haematopoietic+stem/progenitor+cells+in+their+niche.">Live-animal tracking of individual haematopoietic stem/progenitor cells in their niche.</a> Nature. 457 (7225), 92-96 (2009).</li><li>Wang, L., 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=Notch-dependent+repression+of+miR-155+in+the+bone+marrow+niche+regulates+hematopoiesis+in+an+NF-kappaB-dependent+manner.">Notch-dependent repression of miR-155 in the bone marrow niche regulates hematopoiesis in an NF-kappaB-dependent manner.</a> Cell Stem Cell. 15 (1), 51-65 (2014).</li><li>Miyamoto, 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=Myeloid+or+lymphoid+promiscuity+as+a+critical+step+in+hematopoietic+lineage+commitment.">Myeloid or lymphoid promiscuity as a critical step in hematopoietic lineage commitment.</a> Dev Cell. 3 (1), 137-147 (2002).</li><li>Luc, 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=Down+Regulation+of+Mpl+marks+the+transition+to+lymphoid-primed+multipotent+progenitors+with+gradual+loss+of+granulocyte-monocyte+potential.">Down Regulation of Mpl marks the transition to lymphoid-primed multipotent progenitors with gradual loss of granulocyte-monocyte potential.</a> Blood. 111 (7), 3424-3434 (2008).</li><li>Suzuki, M., Moriguchi, T., Ohneda, K., Yamamoto, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+contribution+of+the+Gata1+gene+hematopoietic+enhancer+to+erythroid+differentiation.">Differential contribution of the Gata1 gene hematopoietic enhancer to erythroid differentiation.</a> Mol Cell Biol. 29 (5), 1163-1175 (2009).</li><li>Essers, 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=IFNalpha+activates+dormant+haematopoietic+stem+cells+in+vivo.">IFNalpha activates dormant haematopoietic stem cells in vivo.</a> Nature. 458 (7240), 904-908 (2009).</li><li>Pietras, E. 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=Re-entry+into+quiescence+protects+hematopoietic+stem+cells+from+the+killing+effect+of+chronic+exposure+to+type+I+interferons.">Re-entry into quiescence protects hematopoietic stem cells from the killing effect of chronic exposure to type I interferons.</a> J Exp Med. 211 (2), 245-262 (2014).</li><li>Scott, M. K., Akinduro, O., Lo Celso, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+4-dimensional+tracking+of+hematopoietic+stem+and+progenitor+cells+in+adult+mouse+calvarial+bone+marrow.">In vivo 4-dimensional tracking of hematopoietic stem and progenitor cells in adult mouse calvarial bone marrow.</a> J Vis Exp. (91), e51683 (2014).</li></ol>

Denne protokol beskriver en eksperimentelle design, optimeret til at studere kinetik af hæmatopoietisk celler engraftment af Intravital fluorescerende mikroskopi. I denne undersøgelse, var udvidelsen af myeloide stamceller i en WT BM eller i et hak signalering defekte BM spores i ben calvarium af følgende Lys-normal god landbrugspraksis positive myeloide celler efter BMT i RBPJWT eller RBPJKO modtagere. Denne tilgang er foreslået som en model, der kan anvendes til løsning af lignende spørgsmål, for eksempel: Jeg) ...

Intravital multiphoton Fluorescens mikroskopi (IVFM) er en kraftfuld billedbehandling teknik, der giver mulighed for en høj opløsning, real-time imaging af væv med dybde op til 1mm, afhængigt af væv. Når de påføres mus calvarium, tillader det, observere adfærd af den hæmatopoietisk celler i BM i en ikke-invasiv måde op til 60-100 μm11. Denne fremgangsmåde bruges her til at bestemme kinetik af engraftment af normale myeloide ophav i BM af RBPJ knock-out mus mangler kanoniske Notch signalering.
Seneste arbejde fra vores gruppe viste, at defekte kanoniske Notch signalering i BM mikromiljø fører til en myeloproliferative-lignende sygdom12. Tab af Notch signalering blev opnået ved betinget sletning af DNA bindende domæne af RBPJ, kritisk transkriptionsfaktor nedstrøms af canonical Notch signalering, ved hjælp af Mx1-Cre induceret rekombination10. I denne undersøgelse, blev Mx1-Cre/RBPJlox/lox mus model brugt. Betinget sletning af DNA-bindende motiv af RBPJ resulterer i tab af signaler fra alle Notch receptorer. I modellen Mx1-Cre, Cre udtryk er drevet af Mx1 initiativtageren aktiveret ved administration af polyI:C resulterer i induktion af målrettede gen sletning i blod celler samt som stromale komponenter af flere organer, herunder BM, milten og leveren.
Mx1-Cre+/RBPJlox/lox og Mx1-Cre-/RBPJlox/lox mus induceret med polyI:C (hereon angivet som RBPJKO og RBPJWT, henholdsvis) var lethally bestrålet og transplanteret med normale, vildtype hæmatopoietisk celler. Starter fra uge 4 efter transplantation, udviklet RBPJKO modtagere betydeligt leukocytose efterfulgt af splenomegali. Selvom RBPJKO mus præsenteret øget procentdel af myeloide stamceller i BM i uge 8 efter transplantation og på senere tidspunkter, afsløre analyse af BM uger 4 og 6 ikke slående forskelle i deres myeloide celleindholdet i forhold til kontrol RBPJWT modtagere. Denne observation, sammen med det faktum, at Mx1-Cre kommer til udtryk i forskellige hæmatopoietisk organer, rejste spørgsmålet om, hvorvidt BM mikromiljø havde direkte indflydelse på iværksættelsen af myeloproliferative fænotype.
For at afgøre, hvorvidt BM er et kritisk første stedet for udvikling af sygdom, var IVFM af musen calvarium bruges i kombination med BM transplantation (BMT), den RBPJ knock-out model, og en slægt tracking system. Transgene mus at udtrykke EGFP under kontrol af specifikke lysozym promoter (Lys-NGL)9 blev brugt til at opnå donor celler, der kunne visualiseres under BM imaging efter BMT. Lysozym udtryk er specifikke for myeloide celler og Lys-normal god landbrugspraksis markerer celler fra den fælles myeloide stamceller (CMP) til modne granulocyt13.
IVFM af BM på forskellige tidspunkter viste, at Lys-NGL celler homed ligeledes til BM af RBPJWT og RBPJKO modtagere, men udvidet og aflægger hurtigere i BM af RBPJKO modtagere. Denne forskel var dramatisk for den tidligere tidspunkt (uge 2) og faldt over tid (uger 4 og 6). Men disse senere tidspunkt punkter, evaluering af hæmatopoietisk rum i den samme modtager viste en støt stigning i antallet af myeloide celler cirkulerer i PB og lokaliseret i milten af RBPJKO mus, der angiver en øget produktion af celler fra BM i omløb. Analyse af Lys-NGL celler lokalisering i BM af transplanterede mus på 6 uger, viste, at myeloide celler var bosat længere fra Vaskulaturen RBPJKO mikromiljø end i kontrolelementet.
Kollektivt, givet kombinationen af IVFM med disse specifikke dyremodeller indsigt i engraftment dynamikken af myeloide celler i RBPJKO BM mikromiljø. Eksperimentelle design og kvantitativ metode beskrevet her er foreslået som et paradigme, der kan anvendes for at løse lignende spørgsmål. For eksempel, brugen af andre celle specifikke lineage tracking modeller, såsom RAG1-NGL14 eller Gata1-NGL15 mus, kan tillade efter opførsel af lymfoide eller erythroid progenitorceller, henholdsvis i BM.

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combining intravital fluorescent microscopy (ivfm) with genetic models to study engraftment dynamics of hematopoietic cells to bone marrow niches

Stadig flere beviser viser, at normal bloddannelsen er reguleret af særskilte microenvironmental stikord i BM, som omfatter specialiserede cellulære nicher modulerende kritiske hæmatopoietisk stamcelle (HSC) funktioner1,2. Faktisk, et mere detaljeret billede af den hæmatopoietisk mikromiljø nu fremstår, hvori den knoglens endosteale og i endothelial nicher danne funktionelle enheder til regulering af normale HSC og deres afkom3,4,5 . Nye undersøgelser har afsløret betydningen af perivascular celler, adipocytter og neuronale celler i vedligeholdelse og regulering af HSC funktion6,7,8. Desuden er der bevis for, at celler fra forskellige slægter, dvs myeloid og lymfoide celler, hjem og opholde sig i særlige nicher inden for BM mikromiljø. En fuldstændig kortlægning af BM mikromiljø og ombordværende er imidlertid stadig i gang.
Transgene mus stammer udtryk for lineage specifikke fluorescerende markører eller musene genetisk manipuleret til at mangle valgte molekyler i bestemte celler i BM niche er nu tilgængelige. Knock-out og afstamning tracking modeller i kombination med transplantation tilgange, giver mulighed for at forfine viden om specifikke "niche" rolle celler for definerede hæmatopoietisk populationer, som HSC, B-celler, T-celler, myeloide celler og erythroid celler. Denne strategi kan yderligere forstærkede ved at kombinere brugen af to-foton mikroskopi af calvarium. Ved at give i vivo høj opløsning imaging og 3D-rendering af BM calvarium, kan vi nu fastslå netop det sted, hvor specifikke hæmatopoietisk delmængder hjem i BM og evaluere kinetik af deres ekspansion over tid. Her, er Lys-normal god landbrugspraksis Transgene mus (mærkning myeloide celler)9 og RBPJ knock-out mus (mangler kanoniske Notch signalering)10 brugt i kombination med IVFM til at bestemme engraftment af myeloide celler til et hak defekte BM mikromiljø.

Kohorter af 2 RBPJKO og 2 RBPJWT modtagere blev afbildet i en individuel imaging session på forskellige tidspunkter: 24 og 2, 4 og 6 uger efter transplantation af BM Lys-NGL celler (workflow er illustreret i figur 1A).
I hvert mus blev billeder erhvervet fra 6 standard regioner i BM calvarium, identificeret ved deres holdning til tvedeling af den centrale vene (figur 2A, en) og...

Watch this Scientific Journal Video about Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches at JoVE.com

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital Fluorescens mikroskopi (IVFM) af calvarium anvendes i kombination med genetisk dyremodeller for at studere målsøgende og engraftment hæmatopoietisk celler i knoglemarven (BM) nicher.

Kombinerer Intravital fluorescerende mikroskopi (IVFM) med genetiske modeller at studere Engraftment dynamikken i hæmatopoietisk celler til knoglemarven nicher

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Research

JoVE Journal

Developmental Biology

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Automated Quantification of Hematopoietic Cell &#8211; Stromal Cell Interactions in Histological Images of Undecalcified Bone

Automated Quantification of Hematopoietic Cell ÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“ Stromal Cell Interactions in Histological Images of Undecalcified Bone

Automatiseret Kvantificering af hæmatopoietisk celle - stromacelle Interaktioner i Histologiske Billeder af afkalkede Bone

Confocal microscopy is the method of choice for the analysis of localization of multiple cell types within complex tissues such as the bone marrow. ...

An Enzymatic Method to Rescue Mesenchymal Stem Cells from Clotted Bone Marrow Samples

En enzymatisk metode til at redde mesenchymale stamceller fra Clotted knoglemarvsprøver

Mesenchymal stem cells (MSCs) - usually obtained from bone marrow - often require expansion culture. Our protocol uses clinical grade urokinase to degrade ...

Isolating Mesangiogenic Progenitor Cells (MPCs) from Human Bone Marrow

Isolating Mesangiogenic Progenitor Cells MPCs from Human Bone Marrow

Isolering Mesangiogenic progenitorceller (MPCS) fra human knoglemarv

In a research study aimed to isolate human bone marrow (hBM)-derived Mesenchymal Stromal Cells (MSCs) for clinical applications, we identified a novel ...

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

In vitro og in vivo modeller til at studere hornhindeendothel-mesenkymale Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

A RANKL-based Osteoclast Culture Assay of Mouse Bone Marrow to Investigate the Role of mTORC1 in Osteoclast Formation

En RANKL-baserede osteoklast kultur Assay af musen knoglemarven til at undersøge rollen, som mTORC1 i osteoklast dannelse

Osteoclasts are unique bone-resorbing cells that differentiate from the monocyte/macrophage lineage of bone marrow. Dysfunction of osteoclasts may result ...

Isolation, Culture, and Differentiation of Bone Marrow Stromal Cells and Osteoclast Progenitors from Mice

Isolation, kultur og differentiering af knoglemarven stromale celler og osteoklast ophav fra mus

Bone marrow stromal cells (BMSCs) constitute a cell population routinely used as a representation of mesenchymal stem cells in vitro. They reside within ...

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

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 ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Voksen zebrafisk skade modeller at studere virkningerne af Prednisolon i regenererende knoglevæv

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

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 af oxidative skader i den primære mus okulære overfladeceller / stamceller som reaktion på Ultraviolet-C (UV-C) Skader

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

Determination of Self- and Inter-(in)compatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Determination of Self- and Inter-incompatibility Relationships in Apricot Combining Hand-Pollination, Microscopy and Genetic Analyses

Bestemmelse af selv- og inter-(in)kompatibilitetsforhold i abrikos, der kombinerer håndbestøvning, mikroskopi og genetiske analyser

Self-incompatibility in Rosaceae is determined by a Gametophytic Self-Incompatibility System (GSI) that is mainly controlled by the multiallelic locus S. ...