1. Embrione rimozione e dissezione placentare <ol><li> Per cominciare, gli embrioni devono essere prima isolati da dighe incinta. </li><li> Gli animali sono l&#39;eutanasia secondo procedure approvate - nel nostro caso abbiamo intenzione di utilizzare una dose letale di anestetico isoflurano </li><li> In primo luogo, spruzzare il ventre con etanolo e fare una piccola incisione con un paio di forbici. </li><li> Strappare via la pelle con entrambe le mani per scoprire l&#39;addome e tagliare aprire il peritoneo. </li><li> Con una pinza, raccogliere le corna uterine e alla loro raccolta con le forbici ad ogni estremità distale. Mettere le corna uterine in una capsula di Petri riempite con PBS e posto sul ghiaccio. Inoltre, ricordarsi di lavare più volte con PBS prima di isolare la placenta. Ora possiamo isolare la placenta. </li><li> Con due pinze, iniziare con attenzione peeling via il tessuto endometriale che circonda la conceptuses embrione. Si dovrebbe fare attenzione a non forare o comunque infliggere danni strutturali agli embrioni, in modo da complicare la dissezione passi successivi. </li><li> Posizionare il conceptuses isolato in un nuovo piatto di Petri con PBS su ghiaccio e continuare fino a quando tutti conceptuses sono stati liberati dalla endometrio. </li><li> Trasferire una concepito per un nuovo piatto di Petri con PBS posta sotto il microscopio e con due pinze sbucciare la decidua lontano dalla placenta. Rimuovere la maggior quantità di decidua il più possibile le cellule decidua interferirà con cella singola sospensione e citometria a flusso. Una fluidità del movimento continuo peeling è consigliata per ottenere i migliori risultati. </li><li> Tagliare il sacco vitellino lontano dalla placenta alla confluenza tra i due organi e tirare delicatamente il sacco vitellino ed i vasi vitellino lontano dalla placenta. Si deve prestare attenzione a non disturbare il piatto coriali della placenta, in particolare con embrioni precoci. </li><li> Con attenzione tenendo il piatto coriale della placenta con un paio di pinze e il cordone ombelicale con un altro, tirare il cordone ombelicale attaccato e embrione di distanza dalla placenta. </li><li> Rimuovere l&#39;eccesso di tessuto a cellule giganti ai bordi della placenta al fine di minimizzare aggregazione delle cellule durante la preparazione della cella singola sospensione. </li><li> Raccogliere la placenta in un contenitore adeguato, come un tubo da 15 ml Falcon, con% FCS PBS +5 e sul ghiaccio. Fate questo per tutte le placente sezionato. </li><li> A questo punto, è possibile procedere con la preparazione di una cella singola sospensione per citometria a flusso, test funzionali, come la cultura in vitro o il trapianto, o preparare tutta la placenta per la fissazione dei tessuti e fissi blocco congelato embedding per immunoistochimica finale. </li></ol> 2. Preparazione di singole cellule sospensione del tessuto della placenta <ol><li> Noi ora vi mostrano come generare una sospensione singola cellula da placenta per l&#39;analisi FACS. </li><li> Per preparare una sospensione singola cellula da placenta, meccaniche e dissociazione enzimatica è richiesto. In primo luogo preparare una soluzione Collagenasi 0,1% in PBS con 10% FCS e 1% di soluzione di penicillina / streptomicina. </li><li> Aggiungere un volume appropriato di soluzione di collagenasi alla placenta, a seconda di quanti avete. Un volume che è il doppio del volume placenta e tra 2-5ml è raccomandato. </li><li> Utilizzare un ago 16-G montato su una siringa da 5 ml di distruggere meccanicamente i tessuti da passaging la soluzione di collagenasi e la placenta attraverso l&#39;ago 3 volte. Ripetere con un 18-G ago. </li><li> Porre il campione a 37 ° C, 5% CO 2 incubatore per 45 minuti. </li><li> Dopo l&#39;incubazione 45 minuti a collagenasi, il passaggio alla soluzione delle cellule attraverso un ago 20-G, e incubare per altri 45 minuti, in 37 ° C. </li><li> Dopo l&#39;incubazione 1,5 ore in totale collagenasi, il passaggio delle cellule attraverso la soluzione 22-25-G e G aghi 3 volte con ogni ago. </li><li> Filtrare il campione attraverso un filtro da 50 micron collegato a un nuovo tubo da 15 ml Falcon, lavare con PBS +5% FCS e centrifugare a 4 ° C per 5 minuti a 300 × g. </li><li> A questo punto, abbiamo un&#39;unica sospensione cellulare adeguata per l&#39;analisi FACS o test funzionali. </li></ol> 3. Fissazione dei tessuti per le sezioni congelate <ol><li> Immediatamente dopo aver isolato la placenta da ogni embrione, essi sono posti in freddo PBS 1X. Di solito una piastra a 96 pozzetti funziona meglio per placente giovani (E8.5-11.5), ma con i placente, E12.5 e più anziani, a 24 pozzetti può essere più conveniente. </li><li> Ogni placenta viene trasferito in un nuovo pozzo pieno di appena scongelati 4% paraformaldahyde o PFA. Per la E12.5 placenta, il tessuto è spostata in un nuovo pozzo a mano, ma per il 10,5 è possibile rimuovere il PBS con attenzione con una pipetta e aggiungere il PFA. E &#39;importante che la placenta è completamente immerso per 2-4 ore a 4 ° C, a seconda dell&#39;età e delle dimensioni del tessuto. </li><li> Trasferire il tessuto in 30% di saccarosio, o se i tessuti sono molto piccoli e fragili, aspirare il PFA e aggiungere il saccarosio direttamente. In questa fase, la placenta sarà variabile a soluzione. Lasciate che i tessuti pernottamento a 4 ° C.</li><li> Dopo il passo durante la notte nel 30% di saccarosio, i tessuti dovrebbe in ultima analisi, si depositano sul fondo del pozzo, indicativo di crioconservazione corretta. Togliere la metà della soluzione di saccarosio e sostituire il volume con ottobre e il luogo del tessuto indietro nel 4 ° C per 1-2 ore. Questo facilita la penetrazione ottobre </li><li> Trasferire il tessuto al 100% ottobre per 1 ora, dopo di che può essere incorporato. </li></ol> 4. Incorporare il tessuto fissato <ol><li> Stampi di plastica con l&#39;etichetta di identificazione dei tessuti appropriati (ad esempio la muffa show). </li><li> Tirare fuori il tessuto della soluzione ottobre e tagliare a metà la placenta orientando la forma di disco placenta con il lato del cordone ombelicale rivolto verso il basso e con attenzione affettare con una lametta pulita avendo cura di spostare la lama avanti e indietro prima di rilasciare la pressione per assicurare un taglio uniforme. La placenta è tagliata a metà per ottenere il miglior prientation per visualizzare dopo il CSE placentare. </li><li> Mettere la metà placenta al fondo dello stampo con il bordo tagliato a contatto con la superficie inferiore dello stampo. Ripetete l&#39;operazione con un nuovo stampo per l&#39;altra metà. Per 10,5 placente, le due metà può essere messo nello stampo stesso utilizzando la stessa tecnica. </li><li> Quando si versa nello stampo ottobre, può essere difficile mantenere la placenta con l&#39;orientamento corretto. Fissare la placenta in posizione tenendo la metà del tessuto con una pinza. Versare lentamente la soluzione ottobre nello stampo e di evitare di produrre le bolle. Per 10,5 placente, tenere la metà dei tessuti da pinze e il riposo l&#39;altra metà dei tessuti contro l&#39;esterno della pinza. Versare lentamente soluzione ottobre come descritto per E12.5 placente. </li><li> Una volta che lo stampo è riempito di ottobre, subito posto lo stampo su ghiaccio secco, l&#39;ottobre si trasformerà flash bianco il congelamento dei tessuti </li><li> Mettere lo stampo in un sacchetto di plastica contenente Drierite e subito conservare in freezer -80 ° C. </li></ol>

<ol>
	<li>Gekas, C., Dieterlen-Lievre, F., Orkin, S. H., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+placenta+is+a+niche+for+hematopoietic+stem+cells.">The placenta is a niche for hematopoietic stem cells.</a> Dev Cell. 8, 365-375 (2005).</li><li>Rhodes, K. E., Gekas, C., Wang, Y., Lux, C. T., Francis, C. S., Chan, D. N., Conway, S., Orkin, S. H., Yoder, M. C., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emergence+of+Hematopoietic+Stem+Cells+Is+Initiated+in+the+Placental+Vasculature+in+the+Absence+of+Circulation.">The Emergence of Hematopoietic Stem Cells Is Initiated in the Placental Vasculature in the Absence of Circulation.</a> Cell Stem Cell. 2, 252-263 (2008).</li></ol>

Le procedure sperimentali descritte in questo protocollo permetterà per l&#39;isolamento e la visualizzazione del tessuto placentare e staminali ematopoietiche e cellule progenitrici. Per un riepilogo completo di rendimento atteso su cellulare e numero di cellule staminali nella placenta fetale e di altri organi ematopoietici durante lo sviluppo fetale si fa riferimento a Gekas, et al. 2005. Per la localizzazione di cellule staminali e lo sviluppo di altre cellule ematopoietiche nella placenta si fa riferimento a Rodi, et al. 2008.

Elencare le materie: Il glucosio 100mM 10mM NaN3 15-ml tubi (BD) 16-G, 18-G, 20-G, 22-25-G e G aghi (BD) Soluzione di saccarosio 30% 35 millimetri piastre di Petri (BD) 4% paraformaldeide 50 - I filtri delle cellule m (Celltrics) 5-ml siringhe (BD) Fosfatasi alcalina substrato Kit I (vettore rosso, Vector) Fosfatasi alcalina substrato Kit III (vettore blu, Vector) Antibiotici (penicillina / streptomicina, P / S) soluzione Collagenasi di tipo I (Sigma) Cryomold monouso in vinile Stampi campioni (10mm x 10mm x 5 mm, Tissue-Tek) Citocheratina Anticorpo primario (DakoCytomation) Dissezione forcipe, acciaio inox o in titanio, numero 5 o 55 Drierite Siero fetale bovino (FBS) Fisherbrand plastica vetrino da microscopio Mailer (Fisher) Glucosio Ossidasi 1000u/ml Levamisolo (Vector) Montaggio di mezzi, a base acquosa Mounting Medium (VectamountTM AQ) Normale siero di cavallo (Vector) Taglio di temperatura ottimale (ottobre, Tissue-Tek) Perossidasi Kit substrato DAB (Vector) Soluzione tampone fosfato (PBS) con calcio / magnesio (Ca2 + w / Mg2 +) Soluzione tampone fosfato (PBS) senza calcio / magnesio (w / o Ca2 + / Mg2 +) Far scorrere scatola di plastica (detiene il 100 diapositive) Tubi di plastica (180 Nalgene PVC atossico autoclavabile) Polipropilene a fondo tondo Provetta, 5 ml (12x75 mm, BD Falcon) Proteinasi K 20mg/ml Soluzione (Amresco) Prodotti di ricerca International Corp Super Pap Pen HT * Marcatori scorrere (2,5 mm, Fisher) Tessuto-coltura trattata a forma di U (andata e basso) piastre a 96 pozzetti o 24 pozzetti (BD Falcon) Tween20, SigmaUltra (Sigma) Amplificazione Tyramide Kit (Invitrogen) Vectastain ABC fosfatasi alcalina Standard Kit (Vector) Vectastain ABC standard Elite Kit (Vector)

isolation and analysis of hematopoietic stem cells from the placenta

Le cellule staminali ematopoietiche (HSC) hanno la capacità di auto-rinnovarsi e di generare tutti i tipi di cellule dei lignaggi di sangue per tutta la durata di un individuo. Tutte le CSE emergono durante lo sviluppo embrionale, dopo di che le loro dimensioni piscina è gestito da auto-rigenerante divisioni cellulari. Identificare l&#39;origine anatomica di cellule staminali e gli eventi critici dello sviluppo che regolano il processo di sviluppo HSC è stato complicato come molti siti anatomici partecipare durante emopoiesi fetale. Recentemente, abbiamo identificato la placenta come organo principale ematopoietiche dove si generano HSCs e ampliato nel unici nicchie microambientali (Gekas, et al 2005, Rodi, et al 2008). Di conseguenza, la placenta è una fonte importante di cellule staminali durante il loro nascita e l&#39;espansione iniziale. In questo articolo, mostriamo le tecniche di dissezione per l&#39;isolamento di murino placenta da 10,5 e E12.5 embrioni, corrispondenti alle fasi di sviluppo di iniziazione di cellule staminali e il picco nella dimensione del pool di HSC nella placenta, rispettivamente. Inoltre, presentiamo un protocollo ottimizzato per dissociazione enzimatica e meccanica del tessuto placentare in cella singola sospensione per l&#39;uso in citometria a flusso o test funzionali. Abbiamo trovato che l&#39;uso di collagenasi per cella singola sospensione della placenta dà rendimenti sufficiente di cellule staminali. Un fattore importante di rendimento HSC dalla placenta è il grado di dissociazione meccanica prima, e la durata di un trattamento enzimatico. Forniamo anche un protocollo per la preparazione di fisso congelati sezioni di tessuto placentare per la visualizzazione di sviluppare HSCs mediante immunoistochimica nelle loro nicchie precise cellulare. Come ematopoietiche antigeni specifici non vengono mantenute durante la preparazione di sezioni in paraffina, si usano abitualmente fisso sezioni congelate per la localizzazione HSCs placentare e progenitori.

Watch this Scientific Journal Video about Isolation and Analysis of Hematopoietic Stem Cells from the Placenta at JoVE.com

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Abbiamo identificato la placenta come organo principale ematopoietiche durante lo sviluppo. Abbiamo trovato che le cellule staminali ematopoietiche (HSC) sono entrambi generati e ampliato nella placenta in unica nicchie microambientali. Qui, descriviamo le tecniche sperimentali necessari per l&#39;isolamento e la visualizzazione di cellule staminali nella placenta del mouse.

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. ...

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Unit 1

A Hierarchical Stem-Cell System: Blood Cell Formation

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20.3K Views.  University of California, Los Angeles. Abbiamo identificato la placenta come organo principale ematopoietiche durante lo sviluppo. Abbiamo trovato che le cellule staminali ematopoietiche (HSC) sono entrambi generati e ampliato nella placenta in unica nicchie microambientali. Qui, descriviamo le tecniche sperimentali necessari per l'isolamento e la visualizzazione di cellule staminali nella placenta del mouse.

Video: Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

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)

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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 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 ...

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.

In this video, we will demonstrate how to isolate retinal stem cells from the ciliary epithelium of the mouse eye and grow them in culture to form clonal retinal spheres. The spheres that are isolated possess the cardinal properties of stem cells: self-renewal and multipotentiality.

Isolamento di cellule staminali della retina dalla Eye mouse

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of ...

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

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

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

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

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

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

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

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

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

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

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

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 ...

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 ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolamento, caratterizzazione e differenziamento di cellule staminali cardiache dal cuore di topo adulto

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...