This work was supported by the Intramural Research Program of the National Heart, Lung, Blood Institute of the National Institutes of Health. We thank Boris Fehse (University Medical Center Hamburg-Eppendorf, Hamburg, Germany) for providing the five LeGO vector plasmids; Christian A. Combs and Neal S. Young (NHLBI, NIH) for discussions, support and encouragement throughout this study, and Andre LaRochelle (NHLBI, NIH) for assistance with tail vein injections.

Tutti i topi sono stati alloggiati e trattati in conformità con la guida per la cura e l&#39;uso di animali da laboratorio del National Institutes of Health, e iscritti in un protocollo di cura e uso degli animali NHLBI Comitato approvato. Femmina B6.SJL-Ptprc (d) Pep3 (b) / BoyJ (B6.SJL) e C57BL / 6 topi, 6-12 settimane di vita, sono stati utilizzati come donatore e del ricevente, rispettivamente. Un elenco di materiali, reagenti e attrezzature sono fornite nella tabella 1. 1 Lego trasduzione del mouse HSPCs e Trapianto di Midollo Osseo Eseguire le procedure descritte di seguito in un livello di biosicurezza certificata 2 (BSL-2) Armadio (cappa di coltura di tessuti). <ol><li> Produzione di lego vettori di codifica 5 fps <ol><li> Utilizzare il lentivirali &quot;Gene Ontology&quot; (Lego) plasmidi vettori, ognuno che codifica per una diversa FP di un forte promotore SFFV costitutivo, tra cui lego-CER2 (Cerulean), lego-G2 (EGFP), lego-V2 (Venere), lego-T2 (tdTomato), e lego-C2 (mCherry) gentilmente fornito dal Dr. Boris Fehse (University Medical Center Hamburg-Eppendorf, Amburgo, Germania); ora disponibili anche da Addgene 5-7. </li><li> Per produrre vettori LEGO, semi di 5 x 10 6 cellule 293T in 10 piatti cm (utilizzare 12 piatti per ogni vettore) 24 ore prima della trasfezione in 8 ml di I10 supporti (IMDM supplementato con 10% FCS e glutammina / penicillina / streptomicina). </li><li> Trasfezione le cellule con fosfato di calcio con le raccomandazioni del kit CAPHOS leggermente modificati: <ol><li> Per ogni mix piatto 15 mg Lego vettore, 12 mg pCDNA3.HIVgag / pol.4xCTE, 4 mg PREV-TAT, e 1 mg plasmidi pMD2.G-VSV-G in presenza di 50 microlitri 2,5 M CaCl 2, e aggiungere acqua per produrre un volume totale di 500 microlitri. </li><li> Plasmidi di DNA precipitato con 500 ml di 2x HeBS (HEPES-Buffered Saline) e incubare per 20 minuti a temperatura ambiente prima di aggiungere ad ogni piatto di cellule 293T. </li><li> Incubare le cellule per6 ore a 37 ° C 5% di CO 2 incubatore; rimuovere il supporto e aggiungere fresco cultura mediatica 6 ore dopo la trasfezione. </li></ol></li><li> Raccogliere surnatanti di coltura contenente particelle lentivirali da ogni piatto, ogni giorno, per 3 giorni. Ogni collezione surnatante giorno successivo aggiungere media I10 fresco a ogni piatto. <ol><li> Filtrare 0,22 micron pori, piscina e concentrarsi surnatanti di ultracentrifugazione su un rotore SW28 per 2 ore a 71.900 xg e 4 ° C. </li><li> Risospendere il surnatante in 1 ml di StemSpan media. Aliquota le scorte lentivirali in 200 ml e conservare a -80 ° C. </li></ol></li></ol></li><li> Titolazione di particelle lentivirali Determinare il titolo di particelle vettoriali Lego in surnatanti raccolti attraverso la quantificazione di trasduzione unità / ml utilizzando cellule NIH3T3. I titoli permettono di calcolare il volume di ciascun supernatante vettoriale necessaria per provocare la molteplicità relativa desiderata di infezione (MOI) per porta sperimentalecellule. <ol><li> Tavola 5 x 10 4 NIH3T3 cellule per pozzetto (12-pozzetti) in 2 ml di mezzi I10 un giorno prima della titolazione. </li><li> Preparare diluizioni seriali 5-fold di surnatanti vettoriali in mezzi I10 integrato con solfato di protamina (4 mg / ml è 1,000x). </li><li> Rimuovere il supporto dalle cellule, e sostituirlo con 500 ml di diluizioni surnatante contenente l&#39;equivalente di 2, 0,8, 0,32 e 0,128 ml di virus concentrati. </li><li> Determinare il numero (n) di cellule per pozzetto presenti al momento della trasduzione, contando pure un controllo. </li><li> 6 ore dopo la trasduzione, aggiungere 2 ml di media I10 e incubare per 3 giorni a 37 ° C, 5% di CO 2. </li><li> Raccogliere le cellule da ogni pozzetto usando tripsina-EDTA, e determinare la percentuale di cellule fluorescenti (% FP) mediante citometria a flusso. </li><li> Calcolare il titolo come segue, la media dei titoli ottenuti per ciascun bene (diluizione) con una conseguente FP% tra il 1 e il 50%: titolo (particelle vettore / ml) = nx% FP / volume di surnatante vettore (ml). Calcolare la media dei titoli ottenuti per ciascuna diluizione se la FP% è compreso tra 1 e 50%. </li></ol></li><li> Raccolta delle cellule del mouse, Purificazione, trasduzione, e Trapianti (schematicamente illustrato in Figura 1A adattato da Malide et al. 4). <ol><li> Sacrifica topi donatori da una procedura approvata. Raccolta di midollo osseo da anteriore e posteriore degli arti: sezionare omeri, trovarsi femori e tibie, rimuovere completamente tutto muscoli, legamenti e tessuto in eccesso da ossa, tagliare tutta la fine di ogni osso il più vicino possibile al giunto, e lavare il midollo osseo di l&#39;osso utilizzando un ago G 27-0 su una siringa contenente supporti I10. </li><li> Agglomerare le intere cellule di midollo osseo per 10 min a 500 xg, a 4 ° C e lisare i globuli rossi due volte con 50 ml di tampone ACK. Risospendere le cellule in 10 ml di mezzi I10. </li><li> Purificare (Lin-) cellule progenitrici lignaggio negativi con il kit di lignaggio esaurimento MACS secondo un kit Protoco modificatol: <ol><li> Utilizzare 30 ml di microsfere anti-biotina invece di 10 ml / 10 7 cellule; completare le fasi di lavaggio con i media I2 (IMDM integrato con il 2% FCS) al posto del tampone. </li><li> Inserire una fase di lavaggio tra l&#39;incubazione con Cocktail biotina-anticorpo e l&#39;incubazione con anti-biotina microsfere; ed equilibrare la colonna con I10 posto del tampone. </li></ol></li><li> Culture cellule LIN- per 48 ore ad una densità iniziale di 5 x 10 5 cellule / ml in mezzi StemSpan supplementato con 10 ng / ml di IL-3 murino, 100 ng / ml di IL-11 murina, 100 ng / ml umano Flt-3 ligando, e 50 ng / ml fattore delle cellule staminali murine. </li><li> Trasferimento da 1 a 4 x 10 5 cellule a 12 pozzetti piastre rivestite con RetroNectin e trasdurre con surnatanti Lego, ciascuna ad una molteplicità di infezione (MOI) di 6-7 per ottenere il 50% di efficienza di espressione per ogni FP, in presenza di StemSpan media e citochine dettagliati sopra e 4 mg / ml di solfato di protamina. </li><li> Trasdurre cellulecon ogni vettore Lego singolarmente come descritto al punto 1.3.5 e mescolare le cellule dopo trasduzione (chiamato Mix) o co-trasdurre cellule con tutti i surnatanti 5 Lego vettoriali contemporaneamente (chiamato Co). Rimuovere le cellule 24 ore più tardi, lavare una volta con StemSpan media senza citochine, e risospendere in mezzi StemSpan senza citochine per il trapianto, o trasferirli su supporti fresco con citochine, e di cultura per ulteriori 96 ore prima di microscopia confocale e citometria a flusso. <ol><li> Eseguire trapianto di midollo osseo infondendo trasdotte Lin- HSPCs tramite iniezione in vena della coda 11 monodose Gy destinatari pre-irradiati (6-18 ore prima del trapianto) i destinatari, in un volume di 100 l, ad una dose di cellule per topo corrispondente a 1 cellule -4 x 10 5 Lin- originariamente placcato per la trasduzione. In ogni esperimento, trapianto di un&#39;intera coorte di animali con la stessa popolazione di cellule del midollo osseo del donatore trasdotte. </li><li> Per la caratterizzazione in vitro di trasdotte HSPC, la cultura le cellule per ulteriori 4-5 giorni in StemSpan completato con citochine e analizzare mediante citometria a flusso per efficienza di trasduzione, e al microscopio per la diversità di colore. <ol><li> Sacrificio singoli topi e recuperare tessuti e organi per l&#39;imaging, a vari tempo indica fino a 120 giorni post-trapianto. <ol><li> Spruzzare con etanolo la pelliccia del mouse e incidere la pelle sul lato ventrale evitando capelli diffusione. </li><li> Sezionare e accise secondo le procedure standard della milza, linfonodi poplitei, polmoni, cuore, pelle, tessuto adiposo, muscolo scheletrico, rene, fegato, così come la testa di accesso ai calvaria. </li><li> In alternativa, utilizzare calvaria o impostare una finestra ossea per l&#39;imaging dal vivo più volte nel corso del tempo 2,8,9 o immagine organi superficiali come i nodi della pelle e linfonodi in animale vivo. </li></ol></li><li> Mettere singoli organi in 35 millimetri piastre di coltura numero 0 copertura in vetro a 50-10056, l PBS per l&#39;imaging intatti, senza sezionamento, fissazione o ulteriore lavorazione come descritto al punto 2.5. </li><li> Per l&#39;imaging volumetrico, mantenere gli organi in in PBS freddo fino al momento dell&#39;uso. Per time-lapse imaging procedere immediatamente al di imaging di organi in DMEM, 10% FBS contenenti 20 HEPES mM a 37 ° C </li></ol></li></ol></li></ol></li></ol> 2 confocale e microscopia a due fotoni e Image Analysis <ol><li> Eseguire immagini di microscopia utilizzando un sistema a cinque canali confocale e multiphoton Leica SP5 dotato di multi-linea Argon, diodo 561 nm, 594 nm HeNe, e HeNe 633 nm laser visibili. Utilizzare in modalità 2-fotone, un femtosecondi pulsata Ti: zaffiro (Ti-Sa) laser, sintonizzabile per l&#39;eccitazione da 680 a 1.080 nm con correzione dispersione. Utilizzare in esperimenti che coinvolgono rosso-spostato PQ (tdTomato e mCherry), un oscillatore parametrico ottico (OPO) laser per estendere la gamma di lunghezze d&#39;onda di uscita fino a 1,030-1,300 nm, in sequenza o simultaneamente con tegli Ti-Sa laser. </li><li> Immagine vari tessuti utilizzando una HCX-IRAPO-L 25X / 0,95 NA obiettivo immersione acqua (WD = 2,5 mm), a-PLAPO CS HC-20X / 0,70 obiettivo NA secco (WD = 0.6 mm), o HC-PL-IRAPO 40X /1.1 NA obiettivo di immersione in acqua (WD = 0.6 mm). </li><li> Ottenere confocale spettri di fluorescenza di raccolta pile lambda (xyλ) della luce emessa in 5 nm-larghezze di banda dello spettro visibile dalle cellule BM trasdotte con un singolo FP Lego vettore, nonché di controllo, non-trapiantato BM. <ol><li> Immagini di processo con il software Leica LAS v2.4.1-AF per creare spettri di riferimento di tutti i 5 fps, nonché di autofluorescenza (Figura 2A). Utilizzando l&#39;individuo spettri FP e la differenza di luminosità fluorescenza (espressa in% di EGFP) di Cerulean (79%), Venere (156%), tdTomato (283%) e mCherry (47%) impostato 5 canali di imaging in sequenza come segue: Cerulean (458 / 468-482 nm), EGFP (488 / 496-514 nm), Venere (514 / 523-558 nm), tdTomato (561 / 579-597 nm), e mCherry (594 / 618-670 nm) ( <strong&gt; Figura 2B). </li><li> Convalidare separazione accurata PQ, come le cellule trasdotte con ogni singolo FP sono visibili solo nel canale corrispondente e assente dal resto (Figura 2C). Inoltre, questo approccio multi-canale definito crea un &quot;colorprint&quot; uniche a 5 canali che consente la successiva analisi del colore e il monitoraggio clonale e ha il vantaggio di ridurre i tempi di acquisizione rispetto alle immagini spettrali. </li></ol></li><li> Emissione di fluorescenza separato, in modalità 2-fotone, da alta efficienza specchi dicroici personalizzato e raccogliere con un quattro canali non descanned rivelatore (NDD) come segue: uno specchio dicroico a 568 nm seguito da uno specchio dicroico a 465 nm, seguita da un filtro di emissione 452/45 nm per SHG o autofluorescenza e cerulea, un filtro 525/40 nm di emissione per EGFP o Venere, e uno specchio dicroico 648 nm e 620/60 nm filtro delle emissioni per tdTomato o mCherry. <ol><li> Rivelare informazioni strutturali da microscopia a due fotoni isegnale (SHG) da ossa e del collagene fibrillare (eccitato a 920 nm o 860 nm raccolti in una dispersa posteriore imaging contrasto ntrinsic (come descritto sopra) di autofluorescenza dei tessuti da elastina, NADH, (eccitato a 780 nm), e la seconda armonica generati direzione) 10,11. </li></ol></li><li> Per volume rendering 3D, raccogliere le serie di immagini xyz (tipicamente 1 x 1 x 4 micron 3 dimensioni voxel) lungo l&#39;asse z a 5 micron intervalli su una gamma di profondità (150-300 micron) in tutto il midollo osseo e altri tessuti, su vaste regioni utilizzando la funzione piastrella del software per generare automaticamente i volumi cuciti comprendente un intero fossa sterno, circa 2,5 x 1,2 mm 2 (xy) e 300 micron (z) (figure 2D-2E) e Movie 1. <ol><li> Immagine cranica il midollo direttamente attraverso il cranio, senza sezionamento 2, 8. </li><li> Sezione sterno lungo il piano sagittale (bisezione) e poitrasversalmente al giunto tra 2 sternale fossato e immagine attraverso il taglio faccia 3,4,12 (Panoramica Figura 1B adattato da Takaku et al 3). </li></ol></li><li> Per 4D time-lapse imaging, posizionare asportati recentemente poplitea linfonodi, milza, polmone, o campioni calvaria non tagliata da 35 mm piatti della cultura numero 0 coprioggetti, in 50-100 ml DMEM 10% FBS contenente 20 HEPES mM a 37 ° C e utilizzare 2-fotone Ti-Sa di eccitazione (860 nm) combinata con l&#39;eccitazione confocale (458 nm, 488 nm, 514 nm, 561 nm, 594 nm) e Leica scanner di risonanza (8000 Hz / sec), prendendo z-stack (~ 80 micron) ad intervalli di 20 sec per un massimo di 1 ora. <ol><li> Per simultaneo a tre colori impiego di imaging 2P Ti-Sa sintonizzati a 860 nm contemporaneamente con il laser OPO (sintonizzato a 1.130 nm per tdTomato, o 1.140 nm mCherry) (Movie 2). </li></ol></li><li> Ricostruzione e analisi dei volumi 3D e sequenze temporali 4D <ol><li> Utilizzare Imaris x64 softwsono dati di cellule fluorescenti all&#39;interno del midollo osseo e diversi tessuti e di esportarle come filmati 3D-rotazione versione 7.6, o pacchetti software alternativi per trasformare i-pile di tegole di immagini grezzi in volume rendering (3D), come descritto in precedenza 3,4 (Film 1) step-by-step esempio: <ol><li> In Imaris, Aprite l&#39;immagine z-stack; Selezionare &quot;Superare&quot; per visualizzare la visualizzazione volume 3D. </li><li> Utilizzare &quot;navigare&quot; per alzare il volume intorno a 360 °. </li><li> Selezionare &quot;Animazione&quot; per creare un&#39;animazione; utilizzare fotogrammi chiave per aggiungere viste selezionate, zoom, rotazioni del volume; Visualizzare in anteprima il filmato, modificare come necessario e salvare il file in un formato filmato desiderato (es: avi, mov). </li></ol></li><li> Per la valutazione quantitativa delle cellule e di clone di frequenza 3D, posizioni, dimensioni e distribuzione, ulteriore processo gli z-stack utilizzando i moduli Imaris XT che integrano applicazioni MATLAB spettacolo meas distanzamisurazio- (utilizzando un algoritmo di trasformazione distanza) e cluster analisi, come descritto in precedenza 3,4. Trasforma le sequenze di pile di immagini nel corso del tempo in quattro dimensioni (4D) film volumi rendering con il software x64 Imaris, e utilizzare spot e analisi di superficie per il monitoraggio semiautomatico della motilità cellulare in tre dimensioni utilizzando algoritmi di movimento autoregressivi. </li><li> Eseguire l&#39;analisi multispettrale delle cellule come segue: estrarre il multi-colore spot etichettati con l&#39;aggiunta di tutti i 5 canali attraverso il canale aritmetica dei Imaris XT in un nuovo canale; determinare per tutti i punti (celle) l&#39;intensità di fluorescenza media in ciascuno dei canali dei componenti 5. Esportare i valori nel software Excel per tracciare i grafici che mostrano il &quot;colorprint&quot; unico ovvero la relativa% di ogni FP a singola cellula (Figura 2E grafico). Correggere i set di dati per la deriva dei tessuti, e calcolare la velocità delle cellule e lo spostamento nel tempo, le lunghezze delle tracce, percorsi e distanze; I valori di esportazione in Excel software per tracciare grafici. Montare figure composite con Adobe Photoshop software CSS 5. </li></ol></li></ol>

<ol>
	<li>Lo Celso, ., C, D. T., Scadden, <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, 3529-3535 (2011).</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, 92-96 (2009).</li><li>Takaku, 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=Hematopoiesis+in+3+dimensions+human+and+murine+bone+marrow+architecture+visualized+by+confocal+microscopy.">Hematopoiesis in 3 dimensions human and murine bone marrow architecture visualized by confocal microscopy.</a> Blood. 116, e41-e55 (2010).</li><li>Malide, D., Metais, J. Y., Dunbar, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+clonal+analysis+of+murine+hematopoietic+stem+and+progenitor+cells+marked+by+5+fluorescent+proteins+using+confocal+and+multiphoton+microscopy.">Dynamic clonal analysis of murine hematopoietic stem and progenitor cells marked by 5 fluorescent proteins using confocal and multiphoton microscopy.</a> Blood. 120, e105-e116 (2012).</li><li>Weber, K., Mock, U., Petrowitz, B., Bartsch, U., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+gene+ontology+(LeGO)+vectors+equipped+with+novel+drug-selectable+fluorescent+proteins+new+building+blocks+for+cell+marking+and+multi-gene+analysis.">Lentiviral gene ontology (LeGO) vectors equipped with novel drug-selectable fluorescent proteins new building blocks for cell marking and multi-gene analysis.</a> Gene Ther. 17, 511-520 (2010).</li><li>Weber, K., Bartsch, U., Stocking, C., Fehse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+multicolor+panel+of+novel+lentiviral+'gene+ontology'+(LeGO)+vectors+for+functional+gene+analysis.">A multicolor panel of novel lentiviral 'gene ontology' (LeGO) vectors for functional gene analysis.</a> Mol Ther. 16, 698-706 (2008).</li><li>Weber, 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=RGB+marking+facilitates+multicolor+clonal+cell+tracking.">RGB marking facilitates multicolor clonal cell tracking.</a> Nat Med. 17, 504-509 (2011).</li><li>Lo Celso, ., Lin, C. P., 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=In+vivo+imaging+of+transplanted+hematopoietic+stem+and+progenitor+cells+in+mouse+calvarium+bone+marrow.">In vivo imaging of transplanted hematopoietic stem and progenitor cells in mouse calvarium bone marrow.</a> Nature protocols. 6, 1-14 (2011).</li><li>Kohler, 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=Altered+cellular+dynamics+and+endosteal+location+of+aged+early+hematopoietic+progenitor+cells+revealed+by+time-lapse+intravital+imaging+in+long+bones.">Altered cellular dynamics and endosteal location of aged early hematopoietic progenitor cells revealed by time-lapse intravital imaging in long bones.</a> Blood. 114, 290-298 (2009).</li><li>Campagnola, P. 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=Three-dimensional+high-resolution+second-harmonic+generation+imaging+of+endogenous+structural+proteins+in+biological+tissues.">Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues.</a> Biophys J. 82, 493-508 (2002).</li><li>Weigert, R., Sramkova, M., Parente, L., Amornphimoltham, P., Masedunskas, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+microscopy+a+novel+tool+to+study+cell+biology+in+living+animals.">Intravital microscopy a novel tool to study cell biology in living animals.</a> Histochem Cell Biol. 133, 481-491 (2010).</li><li>Zhang, 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=Mouse+models+of+MYH9-related+disease+mutations+in+nonmuscle+myosin+II-A.">Mouse models of MYH9-related disease mutations in nonmuscle myosin II-A.</a> Blood. 119, 238-250 (2012).</li></ol>

Gli autori dichiarano interessi finanziari concorrenti.

Descriviamo qui i dettagli di una potente metodologia recentemente messo a punto per il monitoraggio cellulare clonale, che unisce la grande diversità generata contrassegnando con-5FP codifica vettori Lego e l&#39;imaging confocale con tandem e microscopia a 2 fotoni per ottenere volumetrico e l&#39;imaging dinamico in tessuti vivi combinatorio. Questo ha esteso l&#39;uso della marcatura registrando confocale identità spettrale in 5 &quot;distinti&quot; canali a 8-bit di colore a base di FP. Così il relativo rapporto...

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.
This was corrected in the discussion from:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 30 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.
to:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 300 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.

Erratum: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

La produzione di cellule del sangue, ematopoiesi definito, è gestito da una piccola popolazione di staminali e progenitrici di cellule ematopoietiche (HSPCs). Queste cellule risiedono nel midollo osseo (BM) in una nicchia microambientali complesso costituito da osteoblasti, cellule stromali, tessuto adiposo, e le strutture vascolari, tutti implicati nel controllo di auto-rinnovamento e differenziazione 1,2. Come intatto BM è tradizionalmente inaccessibile alle osservazioni dirette, le interazioni tra HSPC e il loro microambiente rimane in gran parte indefinita in vivo. In precedenza, abbiamo stabilito una metodologia per visualizzare l&#39;architettura 3D della BM intatta utilizzando confocale a fluorescenza e riflessione microscopia a 3. Abbiamo caratterizzato l&#39;espansione di EGFP-marcati HSPCs nel BM, ma l&#39;uso di una sola FP escludessimo analisi di rigenerazione a livello clonale. Molto di recente abbiamo approfittato della lentivirali Gene Ontology (Lego) i vettori che esprimono costitutivamente flproteine ​​uorescenti (FPS) per contrassegnare in modo efficiente le cellule 4,5. Co-trasduzione del HSPC con 3 o 5 vettori genera una tavolozza variegata di colori combinatorie, consentendo il monitoraggio di molteplici singoli cloni HSPC. Contrassegnato HSPC combinato con nuova tecnologia di imaging permesso di tracciare individuale homing HSPC e attecchimento nel BM di topi irradiati. Vettori lego sono stati usati per marcare HSPC con 3 o 5 diversi fps (da Cerulean, eGFP, Venere, tdTomato, e mCherry) e seguire il loro attecchimento nel tempo nel BM mediante microscopia confocale e 2-fotone, consentendo la visualizzazione chiara delle ossa e di altri strutture a matrice, e la relazione di cloni HSPC ai componenti del loro microambiente. HSPC sono stati purificati come le cellule negative lignaggio da C57Bl / 6 topi BM, trasdotte con vettori lego, e reinfuse tramite iniezione vena della coda in topi congenici mielo-ablato. Monitorando grandi volumi del tessuto sterno BM abbiamo visualizzato attecchimento endostale si verificano primaRigenerazione BM. Gruppi di cellule con spettri di colore diversi apparso inizialmente in prossimità all&#39;osso e progredito a livello centrale nel corso del tempo. È interessante notare che, dopo più di 3 settimane il midollo costituito da ammassi clonali macroscopici, suggerendo diffusione della emopoiesi derivati ​​dalle singole HSPCs contiguo nel midollo, invece di ampia diffusione HSPCs attraverso il flusso sanguigno. C&#39;era meno diversità di colore in momenti in ritardo, suggerendo difficoltà di trasduzione a lungo termine ripopolare le cellule con più vettori. Inoltre, HSPCs formata cluster nella milza, un organo responsabile anche per emopoiesi nei topi. Singole celle HPSC-derivati ​​potrebbero essere risolti nel timo, linfonodi, milza, fegato, polmoni, cuore, pelle, muscolo scheletrico, tessuto adiposo, e reni pure. Le immagini 3D possono essere valutati qualitativamente e quantitativamente per apprezzare la distribuzione di cellule con perturbazioni minime dei tessuti. Infine, illustriamod la fattibilità di studi dinamici live in 4D, combinando multifotonica scansione di risonanza e confocale time-lapse imaging. Questa metodologia consente non invasiva alta risoluzione, multidimensionale cellula-destino tracciato di spettralmente contrassegnati cellule popolazioni nella loro architettura 3D intatta, fornendo un potente strumento per lo studio della rigenerazione dei tessuti e della patologia.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Production of viruses</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-Cer2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27338</td>
			<td>Expression of Cerulean</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-G2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">25917</td>
			<td>Expression of eGFP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-V2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27340</td>
			<td>Expression of Venus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-T2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27342</td>
			<td>Expression of tdTomato</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LeGO-C2</td>
			<td style="width:493px;">Addgene</td>
			<td style="width:216px;">27339</td>
			<td>Expression of mCherry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Calciumm Phosphate Transfection Kit</td>
			<td>Sigma</td>
			<td>CAPHOS-1KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Tissue culture dish</td>
			<td style="width:493px;">BD Falcon</td>
			<td style="width:216px;">353003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">IMDM</td>
			<td style="width:493px;">Gibco, Life Technology</td>
			<td style="width:216px;">12440-053</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Fetal Bovine Serum Heat Inactivated</td>
			<td style="width:493px;">Sigma</td>
			<td style="width:216px;">F4135-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Pen Strep Glutamine</td>
			<td style="width:493px;">Gibco, Life Technology</td>
			<td style="width:216px;">10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Centrifuge Tubes</td>
			<td style="width:493px;">Beckman Coulter</td>
			<td style="width:216px;">326823</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">SW28 Ultracentrifuge</td>
			<td style="width:493px;">Beckman Coulter</td>
			<td style="width:216px;">L60</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Millex Syringe Driven Filter Unite (0.22um)</td>
			<td style="width:493px;">Millipore</td>
			<td style="width:216px;">SLGS033SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse cell collection, purification, transduction and transplantation</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">ACK lysing buffer</td>
			<td style="width:493px;">Quality Biologicals Inc.</td>
			<td style="width:216px;">118-156-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Lineage Cell Depletion Kit (mouse)</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-090-858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">LS columns + tubes</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-041-306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Pre-Separation Filters (30um)</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:493px;">StemSpan SFEM serum-free medium for culture and expansion of hematopoietic cells (500mL)</td>
			<td>StemCell Technologies Inc&#160;</td>
			<td>9650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">murine IL-11 CF</td>
			<td>R &#38; D Systems Inc</td>
			<td>418-ML-025/CF</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">recombinant murine SCF</td>
			<td style="width:493px;">RDI Division of Fitzgerald Industries Intl&#160;</td>
			<td>RDI-2503</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">recombinant murine IL-3</td>
			<td>R &#38; D Systems Inc</td>
			<td>403-ML-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">FLT-3 Ligand</td>
			<td>Miltenyi Biotec Inc. USA</td>
			<td>130-096-480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">12-well plates, Costar</td>
			<td style="width:493px;">Corning Inc.</td>
			<td style="width:216px;">3527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Retronectin</td>
			<td style="width:493px;">Takara Bio Inc</td>
			<td style="width:216px;">T100A/B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Protamine Sulfate</td>
			<td style="width:493px;">Sigma</td>
			<td style="width:216px;">P-4020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Confocal and two-photon microscopy</td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">DMEM</td>
			<td style="width:493px;">Lonza</td>
			<td style="width:216px;">12-614F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">1M Hepes</td>
			<td style="width:493px;">Cellgro, Mediatech Inc.</td>
			<td style="width:216px;">25-060-CI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Glass bottom culture dish P35G-0-20-C</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-20-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Glass bottom culture dish P35G-0-14-C</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Nunc Lab-Tek Chambered Coverglass #1 Borosilicate coverglass; 4-well</td>
			<td>Thermo Scientific</td>
			<td>155383</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Leica TCS SP5 AOBS five channels confocal and multi-photon microscope</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Chameleon Vision II -TiSaph laser range 680-1080nm</td>
			<td style="width:493px;">Coherent</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Chameleon Compact OPO laser range 1030-1350nm</td>
			<td style="width:493px;">Coherent</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HCX-IRAPO-L 25x/0.95 NA water dipping objective (WD=2.5 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HC-PLAPO-CS 20x/0.70 NA dry objective (WD=0.6 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HC-PL-IRAPO 40x/1.1NA water immersion objective (WD=0.6 mm)</td>
			<td style="width:493px;">Leica Microsystems</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;"></td>
			<td style="width:493px;"></td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:493px;">Imaris software version 7.6</td>
			<td style="width:493px;">Bitplane</td>
			<td style="width:216px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo clonal tracking of hematopoietic stem and progenitor cells marked by five fluorescent proteins using confocal and multiphoton microscopy

Abbiamo sviluppato e convalidato un marchio metodologia per il monitoraggio clonale di staminali e progenitrici di cellule ematopoietiche (HSPCs) con alta risoluzione spaziale e temporale per studiare in vivo ematopoiesi con il trapianto di midollo osseo modello sperimentale murino fluorescente. Marcatura utilizzando vettori lentivirali codificanti per proteine ​​fluorescenti (PQ) combinatoria genetica ha permesso la mappatura destino cellulare attraverso avanzate di imaging microscopia. Vettori codificano cinque diversi PQ: Cerulean, EGFP, Venere, tdTomato, e mCherry sono stati usati per trasdurre HSPCs contemporaneamente, creando una tavolozza variegata di cellule di colore marcato. Imaging utilizzando confocale / microscopia a due fotoni ibrido consente la valutazione simultanea alta risoluzione delle cellule marcate in modo univoco e la loro progenie in combinazione con componenti strutturali dei tessuti. Volumetrica analisi su grandi aree rivelano che le cellule HSPC-derivate spettralmente codificati possono essere rilevati in modo non invasivo in vari tessuti intatti, tra cui il midollo osseo (BM), Per lunghi periodi di tempo dopo il trapianto. Studi in diretta conciliano multiphoton di video-rate e confocale time-lapse imaging in 4D dimostrano la possibilità di tracciamento cellulare e clonale dinamica in maniera quantitativa.

Whole-mount 3D confocale / 2-fotoni microscopia ricostruzioni del corso sternale tempo il midollo osseo ha rivelato l&#39;attecchimento e l&#39;espansione dei trapiantati co-5fps in un modello con caratteristiche notevoli: cloni apparso chiaramente delineata, omogeneamente contrassegnati con ampia gamma di colori inizialmente e di progresso nel tempo per contenere preferenzialmente cellule principalmente un colore. Installazione di microscopia confocale ed esempi rappresentativi di immagini 5fps-segnato HSPC nel midollo...

Watch this Scientific Journal Video about In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy at JoVE.com

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Combinatorie 5 proteine ​​fluorescenti marcatura di cellule staminali e progenitrici ematopoietiche consente il monitoraggio in vivo clonale tramite confocale e microscopia a due fotoni, fornendo approfondimenti midollo osseo emopoietico architettura durante la rigenerazione. Questo metodo permette di mappatura destino non invasiva di cellule HSPCs derivate spettralmente-codificati nei tessuti intatti per lunghi periodi di tempo dopo il trapianto.

In vivo clonale Monitoraggio di staminali ematopoietiche e cellule progenitrici Contrassegnato da cinque proteine ​​fluorescenti che utilizzano confocale e microscopia Multiphoton

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

in-vivo-clonal-tracking-hematopoietic-stem-progenitor-cells-marked

Research

JoVE Journal

Biology

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

13.1K Views.  NHLBI/NIH.  Combinatorie 5 proteine ​​fluorescenti marcatura di cellule staminali e progenitrici ematopoietiche consente il monitoraggio in vivo clonale tramite confocale e microscopia a due fotoni, fornendo approfondimenti midollo osseo emopoietico architettura durante la rigenerazione. Questo metodo permette di mappatura destino non invasiva di cellule HSPCs derivate spettralmente-codificati nei tessuti intatti per lunghi periodi di tempo dopo il trapianto. 

Video: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolamento e trapianto di cellule staminali emopoietiche (CSE)

Ricerca

Biologia

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no curative treatment available. Recent advances in stem cell biology have generated new hopes for the development of effective cell based therapies to treat these diseases. Transplantation of various types of stem cells labeled with fluorescent proteins into muscles of dystrophic animal models has been used broadly in the field. A non-invasive technique with the capability to track the transplanted cell fate longitudinally can further our ability to evaluate muscle engraftment by transplanted cells more accurately and efficiently.
In the present study, we demonstrate that in vivo fluorescence imaging is a sensitive and reliable method for tracking transplanted GFP (Green Fluorescent Protein)-labeled cells in mouse skeletal muscles. Despite the concern about background due to the use of an external light necessary for excitation of fluorescent protein, we found that by using either nude mouse or eliminating hair with hair removal reagents much of this problem is eliminated. Using a CCD camera, the fluorescent signal can be detected in the tibialis anterior (TA) muscle after injection of 5 x 105 cells from either GFP transgenic mice or eGFP transduced myoblast culture. For more superficial muscles such as the extensor digitorum longus (EDL), injection of fewer cells produces a detectable signal. Signal intensity can be measured and quantified as the number of emitted photons per second in a region of interest (ROI). Since the acquired images show clear boundaries demarcating the engrafted area, the size of the ROI can be measured as well. If the legs are positioned consistently every time, the changes in total number of photons per second per muscle and the size of the ROI reflect the changes in the number of engrafted cells and the size of the engrafted area. Therefore the changes in the same muscle over time are quantifiable. In vivo fluorescent imaging technique has been used primarily to track the growth of tumorogenic cells, our study shows that it is a powerful tool that enables us to track the fate of transplanted stem cells.

Tracking Dynamics of Muscle Engraftment in Small Animals by In Vivo Fluorescent Imaging

We describe an in vivo fluorescence imaging protocol to monitor muscle regeneration by GFP-labeled myoblasts after transplantation into skeletal muscles of both healthy and dystrophic mice. This protocol can be adapted to study muscle regeneration by transplantation of other types of cells and in other muscular conditions as well.

Monitoraggio Dinamica di attecchimento muscolare nei piccoli animali da In Vivo Fluorescente Imaging

Muscular dystrophies are a group of degenerative muscle diseases characterized by progressive loss of contractile muscle cells. Currently, there is no ...

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 (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

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

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentivirali-mediata Knockdown Durante Ex Vivo</emEritropoiesi> di cellule staminali emopoietiche

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

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

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

L&#39;analisi fenotipica e l&#39;isolamento di murini cellule staminali ematopoietiche e Lineage-committed progenitori

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

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Un&#39;interfaccia utente grafica per il monitoraggio assistito della concentrazione di proteine ​​in protrusioni cellulari dinamiche

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

Migliorare l'accuratezza del flusso Valutazione citometrica del potenziale della membrana mitocondriale nelle cellule staminali ematopoietiche e progenitrici attraverso l'inibizione delle pompe di efflusso

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

Here we describe a reliable method to measure mitochondrial mass and membrane potential in ex vivo cultured hematopoietic stem cells and T cells.

Misurazione della massa mitocondriale e del potenziale della membrana nelle cellule staminali ematopoietiche e t-cellule da parte della citometria di flusso

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production ...

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. 
Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

The method presented here summarizes optimized protocols for assessing cellular bioenergetics in non-adherent mouse hematopoietic stem and primitive progenitor cells (HSPCs) using the extracellular flux analyzer to measure the extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of HSPCs in real time.

Valutazione della bioenergetica cellulare in cellule staminali ematopoietiche di topo e progenitrici primitive utilizzando l'analizzatore di flusso extracellulare

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their ...

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.

The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

In vivo clonale Monitoraggio di staminali ematopoietiche e cellule progenitrici Contrassegnato da cinque proteine ​​fluorescenti che utilizzano confocale e microscopia Multiphoton

In vivo clonale Monitoraggio di staminali ematopoietiche e cellule progenitrici Contrassegnato da cinque proteine fluorescenti che utilizzano confocale e microscopia Multiphoton