Ringraziamo Nobuyuki Onai, Hitoshi Takizawa e Markus Manz per i preziosi consigli. Questo lavoro è stato finanziato dalla National Science Foundation svizzero, Lega svizzera contro il cancro e la Fondazione Ticinese per la Ricerca sul Cancro.

1. Preparazione della sospensione di cellule dal midollo osseo <ol><li> Euthanize il mouse e collocare l&#39;animale in una padella inox e spruzzo etanolo al 70% sulla sua all&#39;addome e alla schiena. Raccogliere femore e tibia di zampe posteriori, e della colonna vertebrale. </li><li> Accuratamente rimuovere tutti i residui dei tessuti molli della colonna vertebrale con un paio di forbici punta acuminata e dalle ossa delle gambe con una garza. Ossa conservare in una provetta Falcon da 50 ml con 30 ml di mezzo RPMI contenente il 10% inattivato al calore siero bovino fetale (FBS), supplementato con 50 pM β-mercaptoetanolo, 100 pM MEM Amminoacidi non essenziali, 1 mM MEM Piruvato di sodio, 2 mM L-glutammina, 100 pg / ml di streptomicina e 100 U / ml penicillina. </li><li> Coprire il fondo di una malta precedentemente sterilizzato con filtri sterili. </li><li> Trasferire le ossa nel mortaio e coprire con uno strato di filtri per assemblare una superficie di attrito. </li><li> Preparare un tubo da 50 ml Falcon dotato di un filtro di Falcon. </li><li> Rompete le ossacon l&#39;aiuto di un pestello fino ad ottenere una sospensione omogenea cella. </li><li> Trasferire la sospensione nel tubo. </li><li> Aggiungi al 5 mL malta fresca di mezzo RPMI completo per lavare e raccogliere le cellule rimanenti. </li><li> Ripetere gli ultimi due punti fino a quando le ossa omogeneizzati appaiono chiari. </li><li> Trasferimento e filtrare in un nuovo tubo la sospensione cellulare per eliminare i detriti tessuto rimanente. </li><li> Centrifugare la provetta a 1.500 rpm per 5 minuti e rimuovere con cautela il surnatante tramite aspirazione. Assicurarsi di non disturbare il pellet di cellule. </li></ol> 2. L&#39;analisi fenotipica <ol><li> Risospendere e lavare cellule di singoli topi in 15 ml di PBS e il 2% FBS. </li><li> Centrifugare la provetta a 1.500 rpm per 5 minuti e rimuovere con cautela il surnatante tramite aspirazione. Assicurarsi di non disturbare il pellet. </li><li> Sospendere il pellet in 5 ml di PBS e il 2% FBS e cellule conteggio. </li><li> Trasferire 1 x 10 6 cellule / pozzetto in 96 pozzetti a fondo tondopiastra. </li><li> Centrifugare la piastra a 1.500 rpm per 5 min. </li><li> Preparare una miscela di anticorpi monoclonali diretti contro i seguenti marcatori di superficie. Diluire come indicato in PBS e 2% FBS per colorare le cellule. <ul type="disc"><li> CD4, CD8, CD3, CD45R, CD19, Gr1, Ter119, NK1.1 coniugati con PE-Cy5 1:200 </li><li> c-Kit coniugato APC 1:100 </li><li> Coniugato con biotina 01:50 SCA1 </li><li> Coniugato a FITC 1:50 CD34 </li><li> FcyR coniugato a PE 1:100 </li><li> IL-7R coniugato con PE-Cy7 1:100 </li></ul></li><li> Eliminare il surnatante e aggiungere 50 microlitri di mix anticorpi monoclonali in ciascun pozzetto. Dissociarsi in singole cellule pipettando. </li><li> Incubare le cellule per 20 minuti al buio a 4 ° C. </li><li> Lavare le cellule due volte con 150 microlitri di PBS e il 2% FBS. </li><li> Centrifugare la piastra a 1500 rpm per 5 minuti e scartare il supernatante capovolgendo il piatto. </li><li> Diluire streptavidina in PBS e 2% FBS come indicato per colorare le cellule. <ul type="disc"><li> Streptavidina coniugatogated di APC-Cy7 1:300 </li></ul></li><li> Aggiungere 50 pl della soluzione ad ogni pozzetto. Dissociarsi in sospensione singola cella pipettando. </li><li> Incubare le cellule per 10 minuti al buio a 4 ° C. </li><li> Lavare le cellule due volte con 150 microlitri di PBS e il 2% FBS. </li><li> Centrifugare la piastra a 1500 rpm per 5 min e scartare il surnatante. </li><li> Risospendere le cellule in 100 pl di PBS e il 2% FBS. </li><li> Acquisire campioni FACS. </li></ol> 3. Arricchimento magnetico di c-Kit + Cells <ol><li> Preparare una soluzione tampone contenente PBS, pH 7,2, 0,5% di albumina di siero bovino (BSA), e 2 mM EDTA. </li><li> Risospendere il pellet di cellule di osso zucchine derivato da almeno 10 topi in 1 mL di soluzione tampone contenente c-kit coniugato APC mAb diluito 1:50. </li><li> Incubare le cellule per 20 minuti al buio a 4 ° C. </li><li> Lavare le cellule due volte con 40 mL di soluzione tampone. </li><li> Centrifugare la provetta a 1.500 rpm per 5 minuti e rimuovere con cautela tha surnatante tramite aspirazione. Assicurarsi di non disturbare il pellet. </li><li> Risospendere il pellet cellulare e aggiungere 50 ul di anti-APC microsfere per ciascun topo eutanasia direttamente al pellet. </li><li> Mescolare bene e incubare per 20 minuti al buio a 4 ° C. </li><li> Lavare le cellule aggiungendo 40 ml di soluzione tampone e centrifugare a 1500 rpm per 5 min. Aspirare il surnatante completamente. </li><li> Risospendere il pellet cellulare in multipli di 4 mL di soluzione tampone ogni tre topi eutanasia. </li><li> Inserire MACS colonne LS nel campo magnetico di un separatore adatto MACS. Utilizzare 1 colonna ogni tre topi eutanasia. </li><li> Preparare le colonne risciacquando con 4 mL di soluzione tampone. </li><li> Applicare sospensione cellulare sulle colonne. </li><li> Raccogliere cellule non marcati che attraversano e lavare la colonna con 4 ml di soluzione tampone. Eseguire fasi di lavaggio con l&#39;aggiunta di 4 ml di soluzione tampone tre volte. Aggiungi nuovo buffer quando il serbatoio si svuota colonna. </li><li> Rimuovere le colonne dal separatore e Place ciascuno su 15 mL tubi falcon. </li><li> Pipettare 4 mL di soluzione tampone su ogni colonna. Immediatamente lavare le cellule marcate magneticamente premendo lo stantuffo nella colonna. </li><li> Centrifugare le provette a 1500 rpm per 5 minuti e rimuovere con cautela il surnatante tramite aspirazione. </li><li> Risospendere ogni pellet con 1 mL di soluzione tampone e pellets di gruppo in un unico tubo di 15 ml Falcon. </li></ol> 4. Ordinamento di cellule staminali ematopoietiche e Lineage progenitori impegnati <ol><li> Preparare una miscela di anticorpi monoclonali diretti contro i seguenti marcatori di superficie. Diluire come indicato in 500 pl PBS e il 2% FBS per colorare le cellule. <ul type="disc"><li> CD4, CD8, CD3, CD45R, CD19, Gr1, Ter119, NK1.1 coniugati con PE-Cy5 1:200 </li><li> c-Kit coniugato APC 1:100 </li><li> Sca-1 coniugato a biotina 1:50 </li><li> Coniugato a FITC 1:50 CD34 </li><li> FcyR coniugato a PE 1:100 </li></ul></li><li> Centrifugare il tubo dal precesezione s a 1.500 rpm per 5 minuti e rimuovere con cautela il surnatante tramite aspirazione. </li><li> Aggiungere la miscela mAbs al pellet. Dissociarsi in sospensione singola cella pipettando. </li><li> Incubare le cellule per 20 minuti al buio a 4 ° C. </li><li> Lavare le cellule due volte con 14mL di PBS e 2% FBS. </li><li> Centrifugare la provetta a 1.500 rpm per 5 min e scartare il surnatante. </li><li> Diluire streptavidina come indicato in 500 pl PBS e il 2% FBS per colorare le cellule. <ul type="disc"><li> Streptavidina coniugata con APC-Cy7 1:300 </li></ul></li><li> Aggiungere la soluzione del tubo. Dissociarsi in sospensione singola cella pipettando. </li><li> Incubare le cellule per 10 minuti al buio a 4 ° C. </li><li> Lavare le cellule due volte con 14 mL di PBS e il 2% FBS. </li><li> Centrifugare la provetta a 1.500 rpm per 5 min e scartare il surnatante. </li><li> Risospendere le cellule in 500 pl di PBS e il 2% FBS. </li><li> Trasferire e filtrare la sospensione cellulare in un nuovo tubo 5 ml per elicessazione definitiva aggregati cellulari che possono interferire con la procedura di ordinamento. </li><li> Aggiungere 5 pl di 7-AAD (usarlo diluito 1:100) alla sospensione cellulare al fine di escludere 7-AAD + cellule morte dalle popolazioni ordinati. </li><li> Ordinare le celle con un FACSAria BD secondo i fenotipi seguenti: <ul type="disc"><li> Cellule LKS: LIN - c-Kit + Sca-1 + </li><li> LT-CSE: LKS CD34 - </li><li> ST-CSE: LKS CD34 + </li><li> PGM: lin - c-Kit + Sca-1 low FcyR alta CD34 + </li><li> Deputati: lin - c-Kit + Sca-1 low FcyR bassa CD34 - </li><li> CMP: lin - c-Kit + Sca-1 low FcyR bassa CD34 + </li></ul></li></ol> 5. Risultati rappresentativi La Figura 1 mostra e con l&#39;esempio della procedura di gating elettronica apunteggio comuni progenitori linfoidi (CLPs) come Lin - / c-Kit Lo / interleuchina (IL)-7R + cellule; Lin - / c-Kit + / Sca-1 + (LKS) e Lin - / c-Kit + / Sca Lo -1 (c-Kit +) delle cellule, dalla c-Kit + gate, comuni progenitori mieloidi (CMP) sono identificati come FcyR Lo cellule CD34 +, progenitori dei granulociti / monociti (BPF) come FcyR hi cellule CD34 + e megacariociti / progenitori eritrocitari (MEP) come FcyR lo CD34 - cellule; dal cancello LKS, LT-HSC e ST-HSC sono identificati come CD34 - CD34 + e cellule, rispettivamente. Il midollo osseo sospensione cellulare può essere arricchito per c-kit + cellule con perline magnetiche (Fig. 2); LT-HSC e ST-HSC può quindi essere ordinati secondo CD34 espressione nonché altri progenitori secondo il fenotipo sopra descritto. CSE può essere ana<html> lisati in microscopia confocale immagini in diretta come mostrato nella figura 4, dove CD34 - cellule sono state colorate con il nucleotide-binding composto chinacrina 11 nonché rosso nucleare (DRAQ5). In CSE, ma non GMP, chinacrina-positivi vescicole sono visibili nel citoplasma 12. Inoltre, CSE ordinati possono essere utilizzati in esperimenti funzionali come mostrato in figura 5, che mostra l&#39;aumento citosolico Ca 2 + in LT-HSC mediante attivazione dei recettori purinergici P2 per esposizione a-adenosina trifosfato (ATP) e ionomicina 12. <img alt="Figura 1" src="/files/ftp_upload/3736/3736fig1.jpg" /> Figura 1. Rappresentante analisi dot plot di Lin-BM cellule. Procedura di gating elettronico a citofluorimetro per identificare e segnare progenitori ematopoietiche e CSE di sospensione di cellule del midollo osseo macchiato come descritto nel testo protocollo. pload/3736/3736fig2.jpg &quot;/&gt; Figura 2. Un sistema per l&#39;arricchimento selettivo di c-Kit + cellule dal midollo osseo. <img alt="Figura 3" src="/files/ftp_upload/3736/3736fig3.jpg" /> Figura 3 analisi del ciclo cellulare di elettronica LKS gated CD34 -. CSE nei controlli sani e nei topi con malattia infiammatoria intestinale colorati con anticorpo Ki-67 e DAPI. Le cellule sono state fissate e permeabilizzate con Lyse / Fix e Perm buffer seguita da colorazione con coniugato FITC Ki-67 e DAPI a 1 mg / ml. Cellule in bicicletta sono a malapena se non del tutto rilevabile nel LKS CD34 - vano HSCs di 12 controlli sani. <img alt="Figura 4" src="/files/ftp_upload/3736/3736fig4.jpg" /> . Figura 4 live microscopia confocale imaging di FACS ordinati CD34 - CSE BPF e colorati con il nucleotide-binding quinacrine composto e nucleare rosso (DRAQ5). I quadranti di piccole dimensioni mostrano pos quinacrinevescicole itive rilevata nel citoplasma di cellule staminali, ma non GMP. <img alt="Figura 5" src="/files/ftp_upload/3736/3736fig5.jpg" /> Figura 5. Citosolico Ca 2 + elevazioni in CSE ordinati caricate con Fura-2 e stimolate con ATP e ionomicina. Tracce da singole cellule vengono visualizzati. Tutte le cellule sono sensibili alle ATP extracellulare che mostravano un aumento di rilievo nel citosolico Ca 2 + dopo l&#39;aggiunta di ATP.

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Il metodo qui descritto consente l&#39;analisi rapida ed accurata di ematopoiesi in topi individuali (Figura 1). Questa analisi in diversi contesti sperimentali, inclusi i modelli murini di infiammazione, autoimmunità, immunodeficienza, malattie degenerative, malattie metaboliche e tumori, consente di indirizzare l&#39;impatto delle condizioni patologiche su ematopoiesi. La Figura 3 mostra l&#39;analisi di attività delle cellule ciclismo su LKS elettronicamente gated CD34...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> RPMI 1640 </td><td> Gibco </td><td> 42401 </td></tr><tr><td> MEM NEAA 100X </td><td> Gibco </td><td> 11140 </td></tr><tr><td> Sodio piruvato </td><td> Gibco </td><td> 11360 </td></tr><tr><td> PenStrep </td><td> Gibco </td><td> 15070 </td></tr><tr><td> PBS </td><td> Gibco </td><td> 20012 </td></tr><tr><td> FBS </td><td> Gibco </td><td> 16000 </td></tr><tr><td> Cella Filtro 40 micron </td><td> BD Falcon </td><td> 352340 </td></tr><tr><td> 7-AAD Soluzione colorante </td><td> BD Pharmingen </td><td> 559925 </td></tr><tr><td> Lyse / Fix tampone </td><td> BD Pharmingen </td><td> 558049 </td></tr><tr><td> Perm tampone III </td><td> BD Pharmingen </td><td> 558050 </td> &lt;/ Tr&gt; <tr><td> Ki-67 </td><td> BD Pharmingen </td><td> 556026 </td></tr><tr><td> DAPI </td><td> Invitrogen </td><td> D21490 </td></tr><tr><td> CD4 (GK1.5) </td><td> eBioscience </td><td> 150041 </td></tr><tr><td> CD8 (53-6,7) </td><td> eBioscience </td><td> 150081 </td></tr><tr><td> CD3 (145-2C11) </td><td> eBioscience </td><td> 150031 </td></tr><tr><td> CD45R (RA3-6B2) </td><td> eBioscience </td><td> 150452 </td></tr><tr><td> CD19 (6D5) </td><td> eBioscience </td><td> 150193 </td></tr><tr><td> Gr1 (RB6-8C5) </td><td> eBioscience </td><td> 155931 </td></tr><tr><td> Tre119 (TER-119) </td><td> eBioscience </td><td> 155921 </td></tr><tr><td> NK-1.1 (PK136) </td><td> eBioscience </td><td> 455941 </td></tr><tr><td> c-kit (2B8) </td><td> eBioscience </td><td> 171171 </td></tr><tr><td> Sca-1 (D7) </td><td> eBioscirienza </td><td> 135981 </td></tr><tr><td> CD34 (RAM34) </td><td> eBioscience </td><td> 110341 </td></tr><tr><td> FcyR (2.4G2) </td><td> eBioscience </td><td> 553145 </td></tr><tr><td> Anti-APC microsfere </td><td> Miltenyi Biotec </td><td> 130-090-855 </td></tr><tr><td> LS Colonne </td><td> Miltenyi Biotec </td><td> 130-042-401 </td></tr></tbody></table>

phenotypic analysis and isolation of murine hematopoietic stem cells and lineage-committed progenitors

Il midollo osseo è il luogo principale in cui CSE e più mature cellule progenitrici del sangue del lignaggio risiedono e di differenziarsi in un organismo adulto. CSE costituiscono una popolazione minuti cellulare delle cellule pluripotenti in grado di generare tutte le linee cellulari del sangue per una vita-time 1. La dissezione molecolare di omeostasi CSE nel midollo osseo ha importanti implicazioni in ematopoiesi, oncologia e medicina rigenerativa. Abbiamo descritto il protocollo etichettatura con anticorpi fluorescenti e la procedura gating elettronico in citometria a flusso per segnare sottoinsiemi progenitrici ematopoietiche e la distribuzione CSE in singoli topi (Fig. 1). Inoltre, si descrive un metodo per arricchire ampiamente progenitori emopoietici, nonché a lungo termine (LT) ed a breve termine (ST) ricostituire CSE da sospensioni di cellule del midollo osseo da un pool di arricchimento magnetico di cellule che esprimono c-Kit. Preparato cellulare risultante può essere utilizzato per ordinare sottoinsiemi selezionati in vitro ein vivo studi funzionali (Fig. 2). Entrambi gli osteoblasti trabecolari 2,3 e 4 endotelio sinusoidale costituiscono nicchie funzionali di supporto CSE nel midollo osseo. Diversi meccanismi nella nicchia osteoblastica, tra cui un sottoinsieme di N-caderina + 3 osteoblasti e l&#39;interazione del Tie2 tirosina chinasi del recettore espresso in CSE con il suo angiopoietina-1 ligando 5 concorrono nel determinare quiescenza CSE. &quot;Ibernazione&quot; nel midollo osseo è fondamentale per proteggere HSCs dalla replica e la stanchezza finale sull&#39;attività ciclistica eccessiva 6. Stimoli esogeni che agiscono sulle cellule del sistema immunitario innato, come Toll-like ligandi del recettore 7 e interferone alfa-6 può anche indurre la proliferazione e la differenziazione di cellule staminali progenitori impegnati nel lignaggio. Recentemente, una popolazione di cellule staminali di topo dormienti all&#39;interno del lin - c-Kit + Sca-1 + CD150 + CD48 - CD34 - popolazione è stato descritto 8. Ordinamento delle cellule CD34 sulla base di un&#39;espressione dalla ematopoietiche progenitori arricchita di sospensione cellulare come qui descritto permette l&#39;isolamento di riposo sia auto-rinnovano LT-CSE e CSE ST-9. Una procedura simile basata su deplezione di cellule positive derivazione e smistamento di LT-HSC con CD48 e Flk2 anticorpi è stato precedentemente descritto 10. Nella presente relazione forniamo un protocollo per la caratterizzazione fenotipica ed ex vivo analisi del ciclo delle cellule di progenitori emopoietici, che possono essere utili per il monitoraggio ematopoiesi in diverse condizioni fisiologiche e patologiche. Inoltre, si descrive un procedimento per FACS smistamento CSE, che possono essere utilizzati per definire fattori e meccanismi che regolano l&#39;automantenimento, espansione e la differenziazione in biologia cellulare e saggi di trasduzione del segnale così come per il trapianto.

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

Un metodo per analizzare la distribuzione di progenitori midollari ematopoietiche citometria di flusso e per isolare in modo efficiente altamente purificate cellule staminali ematopoietiche (CSE) è descritto. La procedura di isolamento è essenzialmente basata su un arricchimento magnetico di c-kit + e le cellule di separazione delle cellule per purificare CSE per studi cellulari e molecolari.

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 ...

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Biology

18.6K Views.  Bellinzona (Switzerland). Un metodo per analizzare la distribuzione di progenitori midollari ematopoietiche citometria di flusso e per isolare in modo efficiente altamente purificate cellule staminali ematopoietiche (CSE) è descritto. La procedura di isolamento è essenzialmente basata su un arricchimento magnetico di c-kit + e le cellule di separazione delle cellule per purificare CSE per studi cellulari e molecolari.

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

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

Isolamento e trapianto di cellule staminali emopoietiche (CSE)

Ricerca

Biologia

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Derivazione di cellule staminali ematopoietiche da cellule staminali embrionali murini

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Isolamento e analisi delle cellule staminali ematopoietiche dalla placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

Isolamento delle prime cellule staminali emopoietiche da murini sacco vitellino e AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation, Culture, and Transplantation of Muscle Satellite Cells

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal nuclei in muscle fibers. In adult muscle, satellite cells are normally mitotically quiescent. Following injury, however, satellite cells initiate cellular proliferation to produce myoblasts, their progenies, to mediate the regeneration of muscle. Transplantation of satellite cell-derived myoblasts has been widely studied as a possible therapy for several regenerative diseases including muscular dystrophy, heart failure, and urological dysfunction. Myoblast transplantation into dystrophic skeletal muscle, infarcted heart, and dysfunctioning urinary ducts has shown that engrafted myoblasts can differentiate into muscle fibers in the host tissues and display partial functional improvement in these diseases. Therefore, the development of efficient purification methods of quiescent satellite cells from skeletal muscle, as well as the establishment of satellite cell-derived myoblast cultures and transplantation methods for myoblasts, are essential for understanding the molecular mechanisms behind satellite cell self-renewal, activation, and differentiation. Additionally, the development of cell-based therapies for muscular dystrophy and other regenerative diseases are also dependent upon these factors.

However, current prospective purification methods of quiescent satellite cells require the use of expensive fluorescence-activated cell sorting (FACS) machines. Here, we present a new method for the rapid, economical, and reliable purification of quiescent satellite cells from adult mouse skeletal muscle by enzymatic dissociation followed by magnetic-activated cell sorting (MACS). Following isolation of pure quiescent satellite cells, these cells can be cultured to obtain large numbers of myoblasts after several passages. These freshly isolated quiescent satellite cells or ex vivo expanded myoblasts can be transplanted into cardiotoxin (CTX)-induced regenerating mouse skeletal muscle to examine the contribution of donor-derived cells to regenerating muscle fibers, as well as to satellite cell compartments for the examination of self-renewal activities.

The isolation and culture of a pure population of quiescent satellite cells, a muscle stem cell population, is essential to the understanding of muscle stem cell biology and regeneration, as well as stem cell transplantation for therapies in muscular dystrophy and other degenerative diseases.&#160;

Isolamento, Cultura e trapianto di cellule muscolari satellite

Muscle satellite cells are a stem cell population required for postnatal skeletal muscle development and regeneration, accounting for 2-5% of sublaminal ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

L&#39;infezione retrovirale di Murine staminali embrionali cellulari derivate Cellule embrionali di corpo per l&#39;analisi di differenziazione emopoietiche

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

Identificazione dei fattori chiave di regolazione auto-rinnovamento e differenziazione in cellule ematopoietiche precursori EML da Analysis RNA-sequencing

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

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 ...

Methods Article

L'analisi fenotipica e l'isolamento di murini cellule staminali ematopoietiche e Lineage-committed progenitori