Il lavoro è stato sostenuto in parte dal National Institute of Health (R01GM079688, R21RR024439 e R21GM075838), National Science Foundation (cbet 0.941.055), e la Fondazione MSU.

1. Isolamento di cellule staminali mesenchimali di ratto Cellule staminali mesenchimali sono state isolate da 6-8 settimane vecchio Sprague-Dawley ratto femmina come descritto in precedenza 1, 2. Isolato MSC possono aderire alla superficie di plastica e facilmente espandersi durante coltura in vitro. <ol><li> L&#39;animale è stato messo in una camera di anestesia e anestetizzati per circa cinque minuti. Durante l&#39;anestesia, osservare la velocità di lampeggio, la respirazione e l&#39;attività motoria. Rimuovere l&#39;animale dalla camera subito dopo si ferma l&#39;attività motoria e il tasso di lampeggiante diventa infrequenti. </li><li> Stendere l&#39;animale verso il basso su una stazione di funzionamento e uccidere l&#39;animale da dislocazione cervicale. Tagliare i femori e tibie dagli arti indietro, togliere la pelle ed i muscoli. </li><li> Mettere il femori e tibie sezionato in 70% di isopropanolo per alcuni secondi e trasferimento 1X D-PBS. </li><li> In un armadio biosicurezza, la femori e tibie sono stati trasferiti in un piatto contenente DMEM 10 centimetri. Ogni osso è stato poi tenuto con le pinzette e le due estremità sono state tagliate aperto con una forbice. Inserire un ago 22G di una siringa da 3 ml e riempirlo con DMEM, poi a filo il midollo in una provetta da 50 ml inserendo l&#39;ago di una estremità aperta del tessuto osseo. Ripetere 2 ~ 3 volte per ogni osso. Quando tutte le zucchine sono stati ottenuti, risospendere le cellule e passare la sospensione cellulare attraverso un colino cellula 70μm per rimuovere i detriti delle ossa e degli aggregati del sangue. </li><li> Spin le cellule a 200g, 4 ° C per 5 minuti e rimuovere il surnatante tramite aspirazione. Risospendere le cellule in media 25ml MSC (DMEM contenente 10% FBS e 1% Pen-Strep). Sospensione cellulare 10ml è stato seminato in ciascuna delle piastre cultura 10cm per un totale di due piatti. Tenere i piatti della cultura a 37 ° C e 5% CO 2 incubatore per 1 ~ 2 settimane. Medio è stato cambiato ogni 2 ~ 3 giorni. </li></ol> 2. Arricchimento di MSC Rat Un certo numero di proteine ​​di superficie sono stati utilizzati per arricchire MSC, tra CD54, CD90, CD73, CD105 e CD271 3-5. Nel nostro studio, abbiamo utilizzato CD54 e CD90 come marcatori per arricchire MSC ordinando cellula magnetica. <ol><li> Quando le cellule ha raggiunto circa l&#39;80% confluenza, aspirare il terreno e aggiungere 4 ~ 5 ml di tripsina-EDTA per ogni piatto. Mettere i piatti nuovo al incubatore e incubare per circa 5 minuti per consentire il distacco delle cellule. Una volta che le cellule erano staccati, aggiungere pari quantità di terreno di coltura per inattivare la tripsina. Raccogliere sospensione cellulare in un tubo 15ml e cellule spin down a 200g, 4 ° C per 5 minuti. </li><li> I passi successivi descrivono come arricchire MSC da due marcatori di superficie CD54 e CD90 secondo il manuale per la separazione delle cellule utilizzando BD IMagnet. Pellet cellulare è stato risospeso in tampone di colorazione della cellula (3% di calore FBS inattivato in 1X D-PBS) a 20 milioni di cellule / ml. Biotinilato anticorpi CD54 (0.25μg per milione di cellule) e biotinilato anticorpi CD90 (0.15μg per milione di cellule) è stato aggiunto e mescolato delicatamente con la sospensione cellulare. Dopo incubazione in ghiaccio per 15 minuti, cellule marcate sono state lavate con un volume in eccesso di tampone 1X BD Imag. Le cellule sono state etichettate centrifugare a 200g, 4 ° C per 5 minuti. </li><li> Vortex il BD Imag particelle streptavidina fondo si aggiunge particelle 40μl per ogni 10 milioni di cellule. Mescolare la particella con le cellule etichettate bene e incubare le cellule a 6 ~ 12 ° C per 30 minuti. Questo permette alle particelle streptavidina di legarsi alla biotinilato anti-CD54 e anti-CD90, che è legato alle proteine ​​di superficie CD54 e CD90 rispettivamente. </li><li> Durante il periodo di incubazione, etichettare un fondo rotondo provetta per raccogliere la frazione positiva. Dopo l&#39;incubazione, portare il volume di etichettatura a 20 milioni di cellule / ml con tampone 1X BD Imag e trasferire le cellule etichettati per il positivo-frazione tubo di raccolta. Posizionare il tubo positivo frazione sul IMagnet BD e lasciate stare per 6 minuti, quindi rimuovere il surnatante con una pipetta Pasteur in vetro con tubo positivo-frazione ancora sul IMagnet BD. </li><li> Rimuovere il positivo frazione tubo dalla IMagnet BD e posizionarlo sul ghiaccio. Aggiungere 1ml ghiacciata 1X BD tampone Imag e risospendere le cellule mescolando delicatamente. Posizionare il tubo di nuovo sul IMagnet BD e lasciar stare per 2 ~ 4 minuti. Eliminare il surnatante con una pipetta Pasteur nuovo vetro. Ripetere il lavaggio come descritto in 2.5 ancora una volta. </li><li> Togliere la provetta dal IMagnet BD e risospendere le cellule nel terreno di coltura, proprio seme 75 centimetri 2 fiasco per il mantenimento delle cellule e un piatto 10cm per citometria a flusso. </li></ol> 3. Verifica della espressione di superficie Marker mediante citometria di flusso Analisi di citometria a flusso è stata effettuata per verificare le cellule abbiamo ottenuto esprimere CD54 e CD90. L&#39;HSC marcatore CD45 è stato utilizzato per confermare che il MSC erano impoverito di cellule staminali. <ol><li> Quando le cellule divengono confluenti ~ 80%, trypsinize cellule con tripsina-EDTA e raccogliere le cellule in una provetta da 15 ml. Centrifugare a 200g, 4 ° C per 5 minuti per raccogliere le cellule. </li><li> Aspirare il surnatante e sicellule h con 1X D-PBS una volta. Risospendere le cellule in tampone di colorazione della cellula (1X D-PBS contenente 2% di FBS e sodio azide 0,05%) ad una concentrazione finale di 5 ~ 10 milioni di cellule / ml e mantenere le cellule in ghiaccio. Aliquota 100μl sospensione cellulare in sei provette etichettate (1 solo le celle; 2. Isotipo di controllo IgG2a;.. 3 Controllo Isotipo IgG1, 4 CD45; 5. CD54;.. 6 CD90). </li><li> Aggiungere i controlli isotipo e gli anticorpi primari a concentrazioni appropriate (IgG2a e IgG1: 20μl per milione di cellule; CD45: 0.5μg per milione di cellule; CD54: 0.25μg per milione di cellule; CD90: 0.15μg per milione di cellule) e incubare a 4 ° C per 30 minuti. </li><li> Lavare le cellule con PBS 1X D-due volte e poi risospendere le cellule nel buffer 100μl colorazione delle cellule. Aggiungi SA-PE (streptavidina-ficoeritrina, utilizzare 0.15μg per milione di cellule) e incubare a 4 ° C per 30 minuti al buio. </li><li> Lavare le cellule marcate con 1X D-PBS due volte. Risospendere le cellule in tampone 400μl colorazione delle cellule e trasferimento in un tubo falcon per l&#39;analisi di citometria a flusso. </li></ol> 4. Separazione di singola colonia di cellule staminali mesenchimali derivate MSC è una popolazione eterogenea composta da diverse sottopopolazioni di cellule di forma diversa, il tasso di crescita così come la capacità di differenziazione 6. Tuttavia, tutte le sottopopolazioni esprimere i marcatori MSC conosciuto e quindi non è possibile utilizzare marcatori per separare queste sottopopolazioni. Pertanto, abbiamo applicato cilindri clonazione di separare le diverse sottopopolazioni, che sono colonie formate da singole cellule. <ol><li> Cellule piastra a circa 50 ~ 100 cellule per piatto 10 cm. Incubare a 37 ° C e 5% CO 2 incubatore per 1 ~ 2 settimane. Durante questo periodo, esaminare colonie ben isolate con un microscopio invertito. Una volta che le colonie hanno raggiunto dimensioni abbastanza grandi (meglio più di 100 cellule in ogni colonia), segnare le colonie con un pennarello sul fondo del piatto. Quando prelevare la colonia da marcare, assicurarsi che non ci sono colonie circostanti nei pressi del raccolto uno. </li><li> Aspirare il terreno e lavare il piatto una volta con D-PBS 1X. Prendete un cilindro sterile clonazione con una pinza sterile e delicatamente luogo intorno alla colonia segnato. Ripetere questa operazione fino a quando tutte le colonie ha segnato un cilindro posto sopra la clonazione. Le colonie raccolte dovrebbero essere distanti tra loro come gli altri che ogni cilindro clonazione contiene solo una colonia. </li><li> Aggiungere 100μl tripsina-EDTA per ogni cilindro la clonazione e mettere il piatto indietro l&#39;incubatore per ~ 5 minuti. Dopo 5 minuti, controllare le cellule al microscopio per vedere se sono arrotondamenti. Quando le cellule hanno innalzato, aggiungere pari quantità di terreno di coltura per inattivare la tripsina. Mescolare la sospensione cellulare con una micropipettor 200μl e trasferire la sospensione cellulare per un piatto 60 millimetri contenente 3 ml di media cultura preriscaldata. Etichetta i piatti correttamente e rimetterli in incubatrice. Rilevamento delle modifiche morfologiche nei prossimi giorni. </li></ol> 5. Rappresentante Risultati Secondo il protocollo descritto nella parte per l&#39;isolamento ratto MSC, MSC plastica aderente deve essere visibile il giorno dopo placcatura. Mentre le cellule continuano a proliferare, le cellule confluenti dovrebbe apparire come le cellule mostrato nella Figura 1A. Quando le cellule raggiungono confluenza ~ 80% (Figura 1B), trapianto può essere eseguita. Durante la sottocultura, tripsina-EDTA è stato utilizzato per staccare le cellule e le cellule sollevato sono piccoli e rotondi come mostrato nella Figura 1C. <img src="/files/ftp_upload/1852/1852fig1.jpg" alt="Figura 1" /> Figura 1. Contrasto delle immagini fase di MSC ratto. (A) confluenti MSC. La maggior parte delle cellule sono simili a mandrino o stella-like. (B) MSC circa l&#39;80% confluenza. (C), le cellule revocato dopo tripsinizzazione sono piccoli e rotondi. Una volta che cellule staminali mesenchimali sono arricchite da cell sorting magnetico, citometria a flusso analisi viene effettuata per verificare le espressioni superficie marcatore. Se l&#39;arricchimento è buono, le cellule dovrebbero mostrare colorazione positiva contro la MSC marcatori CD54 e CD90 ma negativo contro il marker CD45 HSC (Figura 2). Isotipo IgG1 controlli IgG2a e sono utilizzati come controlli negativi. <img src="/files/ftp_upload/1852/1852fig2.jpg" alt="Figura 2" /> Figura 2. Analisi di citometria a flusso delle cellule staminali mesenchimali per i marcatori di superficie. Cellule staminali mesenchimali sono stati etichettati con anticorpi contro IgG2a (isotipo di controllo 1), IgG1 (isotipo di controllo 2), CD45, CD54 e CD90. MSC espresso CD54 e CD90 ma non CD45. Quando le cellule sono seminate a propria densità clonale, colonie dovrebbe passare da singole cellule. Figura 3A rappresenta una colonia formata da una sola cellula. Cilindri di clonazione può quindi essere utilizzata per separare le colonie e le cellule derivate dalle colonie possono essere coltivate separatamente. Figura 3B e 3C rappresenta cellule derivate da due singole colonie. Le cellule derivate da colonia 1 sono come mandrino (Figura 3B), mentre le cellule derivate da colonia 2 sono rotondi (Figura 3C). <pclass = &quot;jove_content&quot;&gt; <img src="/files/ftp_upload/1852/1852fig3.jpg" alt="Figura 3" /> Figura 3. Formazione di colonie di cellule staminali mesenchimali e single-colonia di cellule derivate. MSCs colto in colonie clonale densità forma individuale. Queste colonie possono essere separate da cilindri di clonazione e cellule provenienti da colonie diverse possono essere coltivate separatamente. (A) Una colonia rappresentativo formato da MSC quando placcato a densità clonale. (B) Mandrino-come le cellule derivate da una colonia. (C) cellule rotonde derivata da un&#39;altra colonia.

<ol>
	<li>de Hemptinne, I., Vermeiren, C., Maloteaux, J. M., Hermans, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+glial+glutamate+transporters+in+adult+mesenchymal+stem+cells.">Induction of glial glutamate transporters in adult mesenchymal stem cells.</a> J Neurochem. 91, 155-166 (2004).</li><li>Porter, R. M., Huckle, W. R., Goldstein, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+dexamethasone+withdrawal+on+osteoblastic+differentiation+of+bone+marrow+stromal+cells.">Effect of dexamethasone withdrawal on osteoblastic differentiation of bone marrow stromal cells.</a> J Cell Biochem. 90, 13-22 (2003).</li><li>Chamberlain, G., Fox, J., Ashton, B., Middleton, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+mesenchymal+stem+cells:+their+phenotype,+differentiation+capacity,+immunological+features,+and+potential+for+homing.">Concise review: mesenchymal stem cells: their phenotype, differentiation capacity, immunological features, and potential for homing.</a> Stem Cells. 25, 2739-2749 (2007).</li><li>Abdallah, B. M., Kassem, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem+cells:+from+basic+biology+to+clinical+applications.">Human mesenchymal stem cells: from basic biology to clinical applications.</a> Gene Ther. 15, 109-116 (2008).</li><li>Erica, L., Herzog, L. C., Diane, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Krause+Plasticity+of+marrow-derived+stem+cells.">Krause Plasticity of marrow-derived stem cells.</a> Blood. 102, 3483-3493 (2003).</li><li>Colter, D. C., Sekiya, I., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+subpopulation+of+rapidly+self-renewing+and+multipotential+adult+stem+cells+in+colonies+of+human+marrow+stromal+cells.">Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells.</a> Proc Natl Acad Sci U S A. 98, 7841-7845 (2001).</li></ol>

Questo protocollo descrive come isolare ed arricchire MSC. Un metodo per separare la singola colonia di cellule derivate anche incorporato. Ci sono diversi passaggi che sono importanti per una separazione efficace isolamento, l&#39;arricchimento e la colonia. Mentre fate isolamento delle cellule di ratto, si consiglia di filtrare attraverso un colino cellulare o una maglia di nylon sterili di dimensioni simili a sbarazzarsi dei coaguli di sangue e frammenti ossei. Dopo placcatura le cellule durante la notte, molte cellu...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>1X D-PBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14040-133</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>11885-084</td>
<td>&nbsp;</td>
<tr>
<td>Cell strainer</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352350</td>
<td>&nbsp;</td>
<tr>
<td>FBS</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>26140-079</td>
<td>&nbsp;</td>
<tr>
<td>Pen-Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin-EDTA</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>25200-056</td>
<td>&nbsp;</td>
<tr>
<td>BD Imagnet</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>552311</td>
<td>&nbsp;</td>
<tr>
<td>Biotin mouse IgG2a</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>555572</td>
<td>&nbsp;</td>
<tr>
<td>Biotin mouse IgG1</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>555747</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD54</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL054B</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD90</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL005B</td>
<td>&nbsp;</td>
<tr>
<td>Biotin anti-rat CD45</td>
<td>&nbsp;</td>
<td>Cedarlane Labs</td>
<td>CL001B</td>
<td>&nbsp;</td>
<tr>
<td>BD Imag buffer</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>552362</td>
<td>&nbsp;</td>
<tr>
<td>Round-bottom tube</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>352063</td>
<td>&nbsp;</td>
<tr>
<td>Streptavidin particles</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>557812</td>
<td>&nbsp;</td>
<tr>
<td>SA-PE</td>
<td>&nbsp;</td>
<td>R&amp;D systems</td>
<td>F0040</td>
<td>&nbsp;</td>
<tr>
<td>Cloning cylinder</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C2059</td>
<td>&nbsp;</td>		</tbody>
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isolation and enrichment of rat mesenchymal stem cells (mscs) and separation of single-colony derived mscs

Le MSC sono una popolazione di cellule staminali adulte che è una fonte promettente per applicazioni terapeutiche. Queste cellule possono essere isolate dal midollo osseo e può essere facilmente separato dalle cellule staminali ematopoietiche (HSC) a causa della loro adesione plastica. Questo protocollo descrive come isolare cellule staminali mesenchimali di topo femori e tibie. Le cellule isolate sono state ulteriormente arricchite contro due marcatori di superficie CD54 e CD90 MSC ordinando cellula magnetica. Espressione di marcatori di superficie CD54 e CD90 sono stati poi confermati da analisi di citometria a flusso. HSC marcatore CD45 è stato incluso anche per verificare se l&#39;ordine MSCs erano impoverito di cellule staminali. MSC sono naturalmente molto eterogenei. Ci sono sottopopolazioni di cellule che hanno forme diverse, la proliferazione e le capacità di differenziazione. Queste sottopopolazioni tutti esprimono il marker noto MSC e nessun indicatore unico è stato ancora identificato per le diverse sottopopolazioni. Pertanto, un approccio alternativo per separare le diverse sottopopolazioni utilizza bombole di clonazione per separare le singole cellule derivate colonia. Le cellule derivate dal singolo colonie possono essere colti e valutati separatamente.

Watch this Scientific Journal Video about Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs at JoVE.com

Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

Ratto MSC sono stati isolati da femori e delle tibie e poi arricchita da cell sorting magnetico. Cellule ordinati sono stati confermati per l&#39;espressione di marcatori di superficie mediante citometria a flusso. Queste cellule sono state coltivate a densità clonale a formare singole colonie e poi queste colonie erano separate da cilindri di clonazione.

L&#39;isolamento e l&#39;arricchimento delle cellule staminali mesenchimali di ratto (MSC) e la separazione dei Single-colonia di cellule staminali mesenchimali derivate

MSCs are a population of adult stem cells that is a promising source for therapeutic applications. These cells can be isolated from the bone marrow and ...

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Isolation and Enrichment of Rat Mesenchymal Stem Cells (MSCs) and Separation of Single-colony Derived MSCs

28.3K Views.  City of Hope Cancer Center. Ratto MSC sono stati isolati da femori e delle tibie e poi arricchita da cell sorting magnetico. Cellule ordinati sono stati confermati per l'espressione di marcatori di superficie mediante citometria a flusso. Queste cellule sono state coltivate a densità clonale a formare singole colonie e poi queste colonie erano separate da cilindri di clonazione.

Video: Isolation and Enrichment of Rat Mesenchymal Stem Cells MSCs and Separation of Single-colony Derived MSCs

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

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

Isolamento e di espansione degli animali siero gratuito di cordone ombelicale umano derivato cellule stromali mesenchimali (MSC) e cellule endoteliali progenitrici formanti colonia (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation &amp; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Isolamento e caratterizzazione di Hoechst Basso CD45 Negativo Mouse cellule staminali mesenchimali del polmone

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

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 ...

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

In 2001, researchers at the University of California, Los Angeles, described the isolation of a new population of adult stem cells from liposuctioned adipose tissue that they initially termed Processed Lipoaspirate Cells or PLA cells. Since then, these stem cells have been renamed as Adipose-derived Stem Cells or ASCs and have gone on to become one of the most popular adult stem cells populations in the fields of stem cell research and regenerative medicine. Thousands of articles now describe the use of ASCs in a variety of regenerative animal models, including bone regeneration, peripheral nerve repair and cardiovascular engineering. Recent articles have begun to describe the myriad of uses for ASCs in the clinic. The protocol shown in this article outlines the basic procedure for manually and enzymatically isolating ASCs from large amounts of lipoaspirates obtained from cosmetic procedures. This protocol can easily be scaled up or down to accommodate the volume of lipoaspirate and can be adapted to isolate ASCs from fat tissue obtained through abdominoplasties and other similar procedures.

In 2001, researchers at UCLA described the isolation of a population of adult stem cells, termed Adipose-derived Stem Cells or ASCs, from adipose tissue. This article outlines the isolation of ASCs from lipoaspirates using a manual, enzymatic digestion protocol using collagenase.

Isolamento Manuale di cellule staminali derivate da tessuto adiposo da lipoaspirato umani

In 2001, researchers at the University of  California, Los Angeles, described the isolation of a new population of adult  stem cells from liposuctioned ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Isolamento e Cultura della Dental epiteliali cellule staminali del topo adulto Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides the use of bone marrow and umbilical cord MSCs for exogenous cell therapy, there is also increasing interest in the repair and regenerative potential of resident tissue MSCs. Moreover, they likely have a role in normal organ development, and have been attributed roles in disease, particularly those with a fibrotic nature. The main hurdle for the study of these resident tissue MSCs is the lack of a clear marker for the isolation and identification of these cells. The isolation technique described here applies multiple characteristics of lung resident MSCs (L-MSCs). Upon sacrifice of the rats, lungs are removed and rinsed multiple times to remove blood. Following mechanical dissociation by scalpel, the lungs are digested for 2-3 hr using a mix of collagenase type I, neutral protease and DNase type I. The obtained single cell suspension is subsequently washed and layered over density gradient medium (density 1.073 g/ml). After centrifugation, cells from the interphase are washed and plated in culture-treated flasks. Cells are cultured for 4-7 days in physiological 5% O2, 5% CO2 conditions. To deplete fibroblasts (CD146-) and to ensure a population of only L-MSCs (CD146+), positive selection for CD146+ cells is performed through magnetic bead selection. In summary, this procedure reliably produces a population of primary L-MSCs for further in vitro study and manipulation. Because of the nature of the protocol, it can easily be translated to other experimental animal models.

Isolation of CD146+ Resident Lung Mesenchymal Stromal Cells from Rat Lungs

This protocol describes an isolation technique for obtaining primary lung resident mesenchymal stromal cells from rats, through the use of enzymatic digestion, density gradient separation, plastic adherence and CD146+ magnetic bead selection.

Isolamento di CD146<sup&gt; +</sup&gt; cellule residenti del polmone mesenchimali stromali da Rat Polmoni

Mesenchymal stromal cells (MSCs) are increasingly recognized for their therapeutic potential in a wide range of diseases, including lung diseases. Besides ...

Isolation, Proliferation and Differentiation of Rhesus Macaque Adipose-Derived Stem Cells

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) provide a consistent ex vivo cellular system that are functionally like that of in vivo adipocytes. Use of ADSCs in biomedical research allows for cellular investigation of adipose tissue metabolic regulation and function. ADSC differentiation is necessary for adequate adipocyte expansion, and suboptimal differentiation is a major mechanism of adipose dysfunction. Understanding changes in ADSC differentiation is crucial to understanding the development of metabolic dysfunction and disease. The protocols described in this manuscript, when followed, will yield mature adipocytes that can be used for several in vitro functional tests to assess ADSC metabolic function, including but not limited to assays measuring glucose uptake, lipolysis, lipogenesis, and secretion. Rhesus macaques (Macaca mulatta) are physiologically, anatomically, and evolutionarily similar to humans and as such, their tissues and cells have been used extensively in biomedical research and for development of treatments. Here, we describe ADSC isolation using fresh subcutaneous and omental adipose tissue obtained from 4&#8211;9-year old rhesus macaques. Adipose tissue samples are enzymatically digested in collagenase followed by filtration and centrifugation to isolate ADSCs from the stromal vascular fraction. Isolated ADSCs are proliferated in stromal media followed by approximately 14&#8211;21 days of differentiation using a cocktail of 0.5 &#956;g/mL dexamethasone, 0.5 mM isobutyl methylxanthine, and 50 &#956;M indomethacin in stromal media. Mature adipocytes are observed at approximately 14 days of differentiation. In this manuscript, we describe protocols for ADSC isolation, proliferation, and differentiation in vitro. Although, we have focused on ADSCs from rhesus macaque adipose tissue, these protocols can be utilized for adipose tissue obtained from other animals with minimal adjustments.

In this article, we describe isolation of rhesus macaque derived adipose-derived stem cells (ADSCs) using an enzymatic tissue digestion protocol. Next, we describe ADSC proliferation which includes cell detachment, counting and plating. Lastly, ADSC differentiation is described using specific adipogenic inducing agents. Additionally, we describe staining techniques to confirm differentiation.

Isolamento, proliferazione e differenziamento di cellule staminali derivate dall'adiposio del macaco rhesus

Adipose tissue provides a rich and accessible source of multipotent stem cells, which are able to self-renew. These adipose-derived stem cells (ADSCs) ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Isolamento, caratterizzazione e applicazione terapeutica di vescicole extracellulari da cellule staminali mesenchimali umane in coltura

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Isolamento e caratterizzazione di cellule staminali derivate dal tessuto adiposo

Isolation and Identification of Mesenchymal Stem Cells Derived from Adipose Tissue of Sprague Dawley Rats

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in addition to their great capacity for self-renewal, migration, and proliferation. Adipose tissue is one of the least invasive and most accessible sources of mesenchymal cells. It has also been reported to have higher yields compared to other sources, as well as superior immunomodulatory properties. Recently, different procedures for obtaining adult mesenchymal cells from different tissue sources and animal species have been published. After evaluating the criteria of some authors, we standardized a methodology that is applicable to different purposes and easily reproducible. A pool of stromal vascular fraction (SVF) from perirenal and epididymal adipose tissue allowed us to develop primary cultures with optimal morphology and functionality. The cells were observed adhered to the plastic surface for 24 h, and exhibited a fibroblast-like morphology, with prolongations and a tendency to form colonies. Flow cytometry (FC) and immunofluorescence (IF) techniques were used to assess the expression of the membrane markers CD105, CD9, CD63, CD31, and CD34. The ability of adipose-derived stem cells (ASCs) to differentiate into the adipogenic lineage was also assessed using a cocktail of factors (4 &#181;M insulin, 0.5 mM 3-methyl-iso-butyl-xanthine, and 1 &#181;M dexamethasone). After 48 h, a gradual loss of fibroblastoid morphology was observed, and at 12 days, the presence of lipid droplets positive to oil red staining was confirmed. In summary, a procedure is proposed to obtain optimal and functional ASC cultures for application in regenerative medicine.

Autore in primo piano: Isolamento e identificazione di cellule staminali mesenchimali derivate dal tessuto adiposo di ratti Sprague Dawley  

This protocol describes a methodology for isolating and identifying adipose tissue-derived mesenchymal stem cells (MSCs) from Sprague Dawley rats.

Isolamento e identificazione di cellule staminali mesenchimali derivate dal tessuto adiposo di ratti Sprague Dawley

Adult mesenchymal cells have revolutionized molecular and cell biology in recent decades. They can differentiate into different specialized cell types, in ...