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. ...

isolation-and-analysis-of-hematopoietic-stem-cells-from-the-placenta

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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 of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Isolamento di cellule murine embrionali Hemogenic endoteliali

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Ricerca

Biologia dello sviluppo

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Isolamento e l&#39;espansione delle staminali mesenchimali stromali derivate / cellule da placenta umana Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

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 ...

Biologia

Isolation and Characterization of Mesenchymal Stromal Cells from Human Umbilical Cord and Fetal Placenta

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly gained attention because they do not pose any ethical or moral concerns. Current methods to isolate MSCs from UC yield low amounts of cells with variable proliferation potentials. Since UC is an anatomically-complex organ, differences in MSC properties may be due to the differences in the anatomical regions of their isolation. In this study, we first dissected the cord/placenta samples into three discrete anatomical regions: UC, cord-placenta junction (CPJ), and fetal placenta (FP). Second, two distinct zones, cord lining (CL) and Wharton&#39;s jelly (WJ), were separated. The explant culture technique was then used to isolate cells from the four sources. The time required for the primary culture of cells from the explants varied depending on the source of the tissue. Outgrowth of the cells occurred within 3 - 4 days of the CPJ explants, whereas growth was observed after 7 - 10 days and 11 - 14 days from CL/WJ and FP explants, respectively. The isolated cells were adherent to plastic and displayed fibroblastoid morphology and surface markers, such as CD29, CD44, CD73, CD90, and CD105, similarly to bone marrow (BM)-derived MSCs. However, the colony-forming efficiency of the cells varied, with CPJ-MSCs and WJ-MSCs showing higher efficiency than BM-MSCs. MSCs from all four sources differentiated into adipogenic, chondrogenic, and osteogenic lineages, indicating that they were multipotent. CPJ-MSCs differentiated more efficiently in comparison to other MSC sources. These results suggest that the CPJ is the most potent anatomical region and yields a higher number of cells, with greater proliferation and self-renewal capacities in vitro. In conclusion, the comparative analysis of the MSCs from the four sources indicated that CPJ is a more promising source of MSCs for cell therapy, regenerative medicine, and tissue engineering.

Here, we present a protocol for the dissection of human umbilical cord (UC) and fetal placenta sample into cord lining (CL), Wharton&#39;s jelly (WJ), cord-placenta junction (CPJ), and fetal placenta (FP) for the isolation and characterization of mesenchymal stromal cells (MSCs) using the explant culture technique.

Isolamento e caratterizzazione di cellule mesenchimali stromali dal Cordone ombelicale e placenta fetale

The human umbilical cord (UC) and placenta are non-invasive, primitive and abundant sources of mesenchymal stromal cells (MSCs) that have increasingly ...

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Isolamento dei lipidi cellulari Goccioline: due tecniche di purificazione partire da cellule di lievito e placente umane

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Bioingegneria

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

Isolamento di cellule endoteliali progenitrici dal sangue del cordone ombelicale umano

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Isolation of Leukocytes from the Human Maternal-fetal Interface

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua parietalis). This interface is the anatomical site of contact between maternal and fetal tissues; therefore, it is an immunological site of action during pregnancy. Infiltrating leukocytes at the maternal-fetal interface play a central role in implantation, pregnancy maintenance, and timing of delivery. Therefore, phenotypic and functional characterizations of these leukocytes will provide insight into the mechanisms that lead to&#160;pregnancy disorders. Several protocols have been described in order to isolate infiltrating leukocytes from the decidua basalis and decidua parietalis; however, the lack of consistency in the reagents, enzymes, and times of incubation makes it difficult to compare these results. Described herein is&#160;a novel approach that combines the use of gentle mechanical and enzymatic dissociation techniques to preserve the viability and integrity of extracellular and intracellular markers in leukocytes isolated from the human tissues at the maternal-fetal interface. Aside from immunophenotyping, cell culture, and cell sorting, the future applications of this protocol are numerous and varied. Following this protocol, the isolated leukocytes can be used to determine DNA methylation, expression of target genes, in vitro leukocyte functionality (i.e., phagocytosis, cytotoxicity, T-cell proliferation, and plasticity, etc.), and the production of reactive oxygen species at the maternal-fetal interface. Additionally, using the described protocol, this laboratory has been able to describe new and rare leukocytes at the maternal-fetal interface.

Described herein is a protocol to isolate and further study the infiltrating leukocytes of the decidua basalis and decidua parietalis - the human maternal-fetal interface. This protocol maintains the integrity of cell surface markers and yields enough viable cells for downstream applications as proven by flow cytometry analysis.

L&#39;isolamento dei leucociti di Human Interface materno-fetale

Pregnancy is characterized by the infiltration of leukocytes in the reproductive tissues and at the maternal-fetal interface (decidua basalis and decidua ...

Immunologia e infezioni

Isolation of Leukocytes from the Murine Tissues at the Maternal-Fetal Interface

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing fetus. This tolerance is initiated during coitus, established during fecundation and implantation, and maintained throughout pregnancy. Active cellular and molecular mediators of maternal-fetal tolerance are enriched at the site of contact between fetal and maternal tissues, known as the maternal-fetal interface, which includes the placenta and the uterine and decidual tissues. This interface is comprised of stromal cells and infiltrating leukocytes, and their abundance and phenotypic characteristics change over the course of pregnancy. Infiltrating leukocytes at the maternal-fetal interface include neutrophils, macrophages, dendritic cells, mast cells, T cells, B cells, NK cells, and NKT cells that together create the local micro-environment that sustains pregnancy. An imbalance among these cells or any inappropriate alteration in their phenotypes is considered a mechanism of disease in pregnancy. Therefore, the study of leukocytes that infiltrate the maternal-fetal interface is essential in order to elucidate the immune mechanisms that lead to pregnancy-related complications. Described herein is a protocol that uses a combination of gentle mechanical dissociation followed by a robust enzymatic disaggregation with a proteolytic and collagenolytic enzymatic cocktail to isolate the infiltrating leukocytes from the murine tissues at the maternal-fetal interface. This protocol allows for the isolation of high numbers of viable leukocytes (&#62;70%) with sufficiently conserved antigenic and functional properties. Isolated leukocytes can then be analyzed by several techniques, including immunophenotyping, cell sorting, imaging, immunoblotting, mRNA expression, cell culture, and in vitro functional assays such as mixed leukocyte reactions, proliferation, or cytotoxicity assays.

Described herein is a protocol to isolate and analyze the infiltrating leukocytes of tissues at the maternal-fetal interface (uterus, decidua, and placenta) of mice. This protocol maintains the integrity of most cell surface markers and yields enough viable cells for downstream applications including flow cytometry analysis.

L&#39;isolamento dei leucociti di tessuti murini all&#39;interfaccia materno-fetale

Immune tolerance in pregnancy requires that the immune system of the mother undergoes distinctive changes in order to accept and nurture the developing ...

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 ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Isolamento di cellule deciduali umane primarie dalle membrane fetali del termine intempestivo della placenta

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Isolamento e analisi delle cellule staminali ematopoietiche dalla placenta

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Isolamento di cellule deciduali umane primarie dalle membrane fetali del termine intempestivo della placenta