Differenziazione delle cellule staminali embrionali <ol><li> Raccogliere un pallone vicino confluenti di cellule staminali embrionali (ESC) tramite trattamento con tripsina ESC. </li><li> Risospendere le cellule in 5 ml di mezzi di differenziazione e trasferimento in un non-gelatina rivestito T25. Incubare a 37 ° C per 45 minuti. Recuperare il surnatante e raccogliere le cellule tramite centrifugazione. Nota: fibroblasti embrionali di topo (MEF) dalle culture CES collegare facilmente al non-gelatina rivestito T25 e quindi sono esaurite dalla cultura in questa fase placcatura. Se non sei coltura tuo ESC MEF, è possibile saltare il pre-piastra. </li><li> Risospendere MEF-CES impoverito nei media differenziazione a 333.333 cells/50 ml. Questa concentrazione si traduce in 100 ESC/15 ml. </li><li> Utilizzando pipettatore multicanale, piastra CES a 100 mL cells/15 caduta il 15 cm 2 piastre Petri. Con una pipetta a 8 canali, si dovrebbe essere in grado di adattarsi circa 18-22 righe di gocce al piatto (circa 5 ml per piastra). Nota: per questo protocollo, 2-5 15 cm 2 piatti di gocce sarà più che sufficiente e deve cedere 2-5 x 10 6 sei giorni EB cellule derivate. </li><li> Delicatamente capovolgere le piastre per invertire gocce. Incubare a 37 ° C 5% CO 2 per 48 ore. </li><li> Piscina scende di rimestando delicatamente piatti e trasferimento a 15 ml tubo conico. Lavare piatti con 4-6 ml di PBS e aggiungere 15 ml conica stessa. Nota: è possibile piscina fino a cinque piatti goccia pendente. L&#39;EBS crescerà che continuano a differenziare e se sono troppo concentrati tendono a formare grumi di grandi dimensioni. </li><li> Lasciare in pool EB di risolvere per gravità (circa 10 minuti). Aspirare il surnatante senza disturbare EBS. Risospendere delicatamente in media 10 ml Differenziazione e trasferimento a 10 cm non aderente piastra di Petri. </li><li> Inserire la piastra di Petri con agitazione a 50 rpm e incubare a 37 ° C al 5% di CO 2. </li><li> 24 ore più tardi (quattro giorni di differenziazione), piatti vortice di concentrarsi EB nel centro della capsula di Petri. Accuratamente lo scambio del 50% di media (5 ml) con 5 supporti mL Differenziazione fresca. Ritorna alla piastra incubatore per altri due giorni. </li></ol> EB dissociazione e l&#39;infezione con MSCV-HoxB4-IRES-GFP <ol><li> Il giorno sei di differenziazione, il trasferimento di EB a 15 ml tubo conico. Lasciar riposare per gravità. </li><li> Aspirare media e risospendere in 10 ml di PBS. Lasciare EBS per reinsediare per gravità. Aspirare il PBS. </li><li> Aggiungere 250 ml di miscela di enzimi dissociazione e 1 ml di PBS. Incubare a 37 ° C acqua bagno per 20 minuti, a volte vorticoso tubo di mescolare enzimi e EBS. </li><li> Aggiungere 8 ml di enzima senza buffer di dissociazione. </li><li> Triturare miscela utilizzando pipetta da 5 ml fino EB sono completamente dissociati (circa 10 volte; pipettaggio eccessiva aumenterà la morte entro il EB-derivati ​​preparazione delle cellule). <ol><li> Miscela diventerà nuvoloso con cellule come EB dissociano. </li><li> Posizionamento puntale contro la parte inferiore del tubo conico durante la triturazione creerà una forza di taglio che agevolerà notevolmente dissociazione. </li></ol></li><li> Raccogliere le cellule mediante centrifugazione. </li><li> Risospendere EB cellule derivate in 5 ml di 10% IMDM e contare con esclusione blu trypan Nota: Tra quattro giorni e sei giorni di differenziazione, EBS cavitazione che si traduce in una grande quantità di apoptosi e death.Thus cellulare, a sei giorni, verso l&#39;alto di 30% di EB cellule derivate possono macchiare con trypan blu. </li><li> Risospendere le cellule EB-derivati ​​a 100.000 cellule / 2 ml di 10% + IMDM virale surnatante tale che un MOI di 5-10 è achieved.Add solfato di protamina per una concentrazione finale di 8 mg / mL. Nota: Preparare EB abbastanza / virale mix surnatante alla piastra quattro piastre 6-bene (assumendo 2 ml / pozzetto). </li><li> Tavola 2 ml EB / virale mix surnatante per pozzetto di quattro 6 piatti ben pre-placcato con OP9 cellule stroma (vedi sotto). </li><li> Centrifugare piastre a 2500 rpm a temperatura ambiente per 90 minuti. Trasferire le piastre a incubatore a 37 ° C al 5% di CO 2. </li><li> 4-6 ore più tardi, raccolta surnatante dai piatti e raccogliere tutte le cellule potenziale residuo in sospensione tramite centrifugazione. </li><li> Risospendere il pellet (può essere di piccole dimensioni) in 48 mL IMDM 10% + citochine. Dispensare 2 ml / pozzetto di risospensione di originale in quattro a 6 pozzetti in cui è stata effettuata l&#39;infezione ed un surnatante è stato raccolto. Nota: Sempre preparare 10% IMDM + citochine fresco al momento dell&#39;uso. </li><li> Incubare le piastre a 37 ° C al 5% di CO 2 per sette giorni. Colonie dovrebbe essere evidente da quattro giorni dopo l&#39;infezione e di grandi dimensioni e ben formato da sette giorni. Una vigorosa espansione dovrebbe produrre 40-60 colonie / e il giorno sette. </li><li> Su sette giorni dopo l&#39;infezione, raccogliere e piscina tutte le cellule (comprese OP9 stroma) da tutti i pozzetti di trattamento con tripsina. Nota: NON surnatante o PBS lava impiegati durante tripsina treatment.At questo punto della cultura, alcune colonie possono essere solo vagamente aderente e ci sono molte cellule floaTing in suspension.Collect e piscina tutti i lavaggi e surnatante per garantire che non le cellule potenzialmente preziose vengono scartati. </li><li> Risospendere le cellule in 8 IMDM mL fresco del 10% + citochine. Distribuire 2 ml / fiasco in quattro T75 fiaschi. Aggiungere ulteriori 13 ml + 10% citochine IMDM ad ogni pallone. Incubare a 37 ° C al 5% di CO 2 per tre giorni (un totale di dieci giorni dall&#39;infezione). </li></ol> Cultura e placcatura di OP9 stroma <ol><li> Mantenere OP9 stroma secondo protocolli standard nel 20% a-MEM. OP9 cellule cambierà proprietà quando cresciuto fino a confluenza. Dividi sempre al 80% confluenza a non più di 1:03 split. </li><li> 24 ore prima infezione di sei giorni EB cellule derivate con MSCV-HoxB4-IRES-GFP, piastra OP9 25.000 cellule / pozzetto di quattro-sei pozzetti nel 20% a-MEM. </li></ol> Raccolta, il frazionamento e il trapianto <ol><li> Raccogliere le cellule espanse su OP9 stroma per dieci giorni attraverso il trattamento con tripsina. Contare le celle. Nota: NON surnatante o PBS lava impiegata durante il trattamento con tripsina. A questo punto, nella cultura, alcune colonie possono essere solo vagamente aderente e ci sono molte cellule galleggianti in suspension.Collect e piscina tutti i lavaggi e surnatante per garantire che non le cellule potenzialmente preziose vengono scartati. Nota: Una buona espansione dovrebbe produrre tra i 40-50 x 10 6 cellule / originale a 6 pozzetti infetti, per un totale di 160-200 x 10 6 cellule. </li><li> Cellule possono essere frazionate tramite magnetico selezione tallone o FACS secondo tecniche standard, a questo punto nel protocollo. </li><li> Per il trapianto, le cellule fornire via retro-orbitale o iniezione vena della coda per Rag-2/gc destinatari carenti (peso compreso tra 15-22 grammi), sottoposti ad una dose di scissione di 9,25 Gy di irradiazione 2,5 ore. a parte. Nota: è fondamentale che il peso degli animali compresi tra 15-22 grammi al momento della irradiazione. Se sono più grandi di 22 grammi, 9,25 Gy non sarà sufficiente una dose di irradiazione per garantire la ablazione appropriati e le cellule non possono mediare salvataggio da irradiazione letale e / o innestare il comparto ematopoietico. Se il trapianto di grandi animali, la dose di irradiazione appropriati devono essere determinati sperimentalmente. </li><li> Una dose minima di 2-5 x 10 6 cellule / animale è tenuto a garantire soccorso da irradiazione letale. In caso di frazionamento delle cellule, iniettare 2-5 x 10 6 cellule equivalenti (ad esempio se la popolazione di interesse rappresenta il 10% del pool di cellule non frazionata, iniettare 2-5 x 10 5 cellule / animale). Nota: Se l&#39;iniezione&gt; 2 x 10 6 cellule, SEMPRE iniettare via vena della coda per evitare la formazione di teratoma in orbita, che possono risultare quando un numero elevato di cellule vengono consegnati retro-orbitali. </li><li> Mantenere Rag-2 - / - / gc - / - destinatari carente in gabbie in autoclave. A seconda della &quot;pulizia&quot; della struttura animale, post-trapianto gli animali possono richiedere la somministrazione di acqua acidificata o Trimethiprim-Sulfasoxazole (Septra) per le 5 settimane successive radiazioni letali. </li><li> Aspettatevi&gt; GFP 90% + ES-derivati ​​leucociti nel sangue periferico a 3 settimane dopo il trapianto. Attecchimento deve essere multi-lignaggio, anche se i linfociti sono noti al silenzio retrovirali HoxB4-IRES-GFP. </li></ol>

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>C57Bl/6 Rag-2/gamma-c deficient mice</td>
<td>Animal</td>
<td>Taconic Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plate shaker</td>
<td>Tool</td>
<td>VWR</td>
<td>33998-360</td>
<td>Orbital Shaker by Ikaworks</td>
</tr>
<tr>
<td>Multi-channel pipettor</td>
<td>Tool</td>
<td>VWR</td>
<td>15000-174</td>
<td>ePet by BioHit</td>
</tr>
<tr>
<td>ES trypsin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>0.25% Trypsin in PBS</td>
</tr>
<tr>
<td>Standard trypsin</td>
<td>Reagent</td>
<td>Invtrogen</td>
<td>25300-062</td>
<td>0.05% Trypsin-EDTA</td>
</tr>
<tr>
<td>PBS</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Enzyme-free dissociation buffer</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>13151-014</td>
<td>&nbsp;</td>
<tr>
<td>Dissociation enzyme mix</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>500 mg Collagenase IV</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H2126</td>
<td>freeze in 500 uL aliquots and avoid repeated freezing and thawing.</td>
</tr>
<tr>
<td>DNAse</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D4527</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10-017CV</td>
<td>&nbsp;</td>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3369</td>
<td>8 ug/mL</td>
</tr>
<tr>
<td>EB differentiation media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM+cytokines</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>20% anti-MEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>		</tbody>
		</table>
		</div>

derivation of hematopoietic stem cells from murine embryonic stem cells

Una cellula staminale è definito come una cella con la capacità di entrambe le auto-rinnovarsi e di generare più progenie differenziata. Le cellule staminali embrionali (ESC) sono derivate dalla blastocisti dell&#39;embrione precoce e sono pluripotenti in grado differenziativo. Il loro potenziale differenziativo vasta li ha resi oggetto di ricerche molto centrata sulla dedurre il modo di convincere loro di generare tipi di cellule clinicamente utili. La derivazione di successo di cellule staminali ematopoietiche (HSC) da topo CES è stato recentemente compiuto e possono essere visualizzati in questo protocollo video. HSC, probabilmente la popolazione di cellule clinicamente più sfruttati, sono usati per trattare una miriade di tumori ematopoietici e disturbi. Tuttavia, molti pazienti che potrebbero trarre beneficio dalla terapia HSC non hanno accesso ai donatori idonei. ESC potrebbe fornire una fonte alternativa di HSC per questi pazienti. Il seguente protocollo stabilisce una linea di base da cui ESC-HSC possono essere studiati e informare gli sforzi per isolare da HSC umane ESC. In questo protocollo, ESC si differenziano come corpi embrionali (EBS) per 6 giorni nel commercio sierici pre-screening per la differenziazione ottimale ematopoietiche. EBS sono dissociati e poi infettati con HoxB4 retrovirali. Infetti EB cellule derivate sono placcati in OP9 stroma, un stromali del midollo osseo linea cellulare deriva dalla calvaria di M-CSF-/ - topi, e co-coltura in presenza di ematopoiesi promuovere citochine per dieci giorni. Durante questo co-coltura, le cellule infette espandere notevolmente e si traducono nella generazione un pool eterogeneo di 100s di milioni di cellule. Queste cellule possono quindi essere utilizzati per topi salvataggio e ricostituire letale irradiati.

Watch this Scientific Journal Video about Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells at JoVE.com

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Questa dettagli del protocollo la derivazione di cellule staminali ematopoietiche trapiantabili da topo cellule staminali embrionali (ESC) e la loro successiva iniezione in topi irradiati letalmente destinatario. In breve, ESC si differenziano come corpi embrionali, che sono poi infettati con HoxB4 retrovirali e co-coltura con cellule stromali OP9 e citochine ematopoietiche.

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 ...

derivation-hematopoietic-stem-cells-from-murine-embryonic-stem

Research

JoVE Journal

Biology

13.2K Views.  Harvard Medical School. Questa dettagli del protocollo la derivazione di cellule staminali ematopoietiche trapiantabili da topo cellule staminali embrionali (ESC) e la loro successiva iniezione in topi irradiati letalmente destinatario. In breve, ESC si differenziano come corpi embrionali, che sono poi infettati con HoxB4 retrovirali e co-coltura con cellule stromali OP9 e citochine ematopoietiche.

Video: Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Propagazione di staminali embrionali umane (ES) Cellule

Ricerca

Biologia

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

Isolamento e trapianto di cellule staminali emopoietiche (CSE)

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Derivazione di cellule staminali embrionali umane da Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

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. ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

Immunoprecipitazione della cromatina di cellule staminali embrionali umane

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells 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 and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

L&#39;isolamento e la derivazione di cellule embrionali del mouse Germinal

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

Derivazione di cellule staminali trofoblasto mouse da blastocisti

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivazione efficiente dei diritti dell&#39;uomo e precursori cardiaci e cardiomiociti pluripotenti da cellule staminali embrionali umane con induzione piccole molecole

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...