Questo lavoro è finanziato dal NIH / NCI concedere R01CA-118916

1. Estrarre cellule staminali epiteliali da pelle umana <ol><li> Prima di iniziare la procedura di isolare le cellule staminali epiteliali si ha la necessità di preparare i rispettivi media e reagenti (vedi tabella 1). </li><li> Fresco la pelle del cuoio capelluto umano adulto da procedure lifting o biopsia viene raccolto, incubare in FBS DMEM / 10% / dispasi (4 mg / ml) durante la notte a 4 ° C. Incubazione per 2-4 ore a 37 ° C è anche efficace. Pezzi di pelle deve essere una larghezza massima di 1 cm per consentire enzima per penetrare. </li><li> Trasferire la pelle in una capsula di Petri sterile, tirare fuori di ogni capello dalla pelle afferrando il fusto del capello vicino alla superficie della pelle e tirando con fermezza e senza intoppi. Seleziona follicoli in fase telogen in base alla loro morfologia sotto un microscopio da dissezione, tagliare la regione rigonfiamento (Figura 1A) follicoli trasferimento in un tubo da 15 ml sterilizzati. Follicoli anagen può essere utilizzato anche se tagliato in alto a 1 / 3 del follicolo per follicolo pilifero ottenere regione &quot;bulge&quot; (Figura 1B). </li><li> Incubare i frammenti follicolo isolato in una miscela di 0,05% tripsina-EDTA (GIBCO) e Versene (0,53 mM EDTA 4NA, GIBCO) (1:1) per 15-20 minuti a temperatura ambiente agitando periodicamente, aggiungere 4 ml di DMEM + 10 % FBS per fermare la reazione, spin down per 5 min a 800 giri al minuto. In alternativa, follicolo espianti frammento di tessuto può essere collocato in cultura senza tripsina digerire alle cellule dai follicoli singola cultura. </li><li> Gettare il surnatante con cura (salvo ~ 0,2-0,5 mL per evitare di perdere le cellule) e risospendere in 1 ml KCM (media dei cheratinociti), senza FEG. </li></ol> 2. Cultura pile staminali epiteliali <ol><li> Prima di coltura le cellule staminali epiteliali, mitomicina C trattati con 3T3-J2 cellule (cellule di alimentatore) deve essere preparato come segue. 3T3-J2 cellule sono coltivate in DMEM con FBS 10% prima del trattamento. Rimuovere i supporti coltura delle cellule e lavare due volte con PBS, il trattamento delle cellule con 15μg / mL mitomicina C in DMEM senza siero per 2 ore a 37 ° C, 5% CO 2. Rimuovere la mitomicina C contenenti DMEM e lavare due volte con PBS, le cellule sono pronte all&#39;uso. (Mitomicina C trattati con 3T3-J2 cellule possono anche essere preparati in precedenza e conservati a -80 ° C. Scongelare e semi di cellule un giorno prima che la coltura delle cellule staminali epiteliali primarie, incubare a 37 ° C, 5% di CO 2. 150.000-200.000 3T3-J2 cellule per pozzetto della piastra 6-bene è raccomandato. </li><li> Semi isolato cellule dei follicoli dei capelli staminali a partire dal punto 1.5 in mitomicina C trattati con 3T3-J2 cellule KCM senza pernottamento FEG a 37 ° C, 5% di CO 2, cambiata EGF contenenti KCM il giorno successivo. Le cellule sono coltivate a 37 ° C in atmosfera umida contenente il 5% di CO 2. Tutti i cheratinociti sono alimentati con KCM contenente FEG ogni 2 giorni e coltivate per 14-20 giorni. Controllare ogni giorno cellula al microscopio. Capelli cellule staminali del follicolo si forma colonie (Figura 2A). Espianti follicolo si forma escrescenze dopo 10-14 giorni (Figura 2B). </li></ol> 3. Passaggio cellule staminali epiteliali <ol><li> Lavare le cellule con PBS una volta. Rimuovere le cellule 3T3-J2 alimentatore da Versene (pre-riscaldato a RT) del trattamento. Cultura incubare bombati in Versene per 5 minuti, poi agitare delicatamente ed aspirare le cellule di alimentazione. </li><li> Lavare i capelli con le cellule staminali del follicolo sia Versene o PBS una sola volta. </li><li> Aggiungi pre-riscaldato (a 37 ° C, critici) tripsina-EDTA (2X) e incubare a 37 ° C per 7 minuti o più, se necessario, per le cellule staminali dei follicoli dei capelli di staccarsi. Non più di 15 minuti è il migliore. </li><li> Aggiungi KCM w / o FEG per fermare la tripsina, pipettare delicatamente su e giù per disperdere le cellule, spin down a 200g, 5 minuti. </li><li> Risospendere le cellule con KCM senza EGF, il conte e ri-piastra su mitomicina C trattati con 3T3-J2 strato di cellule. </li></ol> 4. Immortalare cellule staminali epiteliali Pile, di cellule staminali adulte, anche, raggiungere senescenza dopo il passaggio di serie e mesi in coltura. Immortalizzazione di cellule staminali primarie è un modo efficace per alleviare questo problema. <ol><li> Piastra primaria cellule staminali epiteliali (~ 350.000 cellule / pozzetto di 6 pozzetti) al passaggio 1 su strato alimentatore (3T3-J2) e la cultura per 2 giorni. </li><li> Cultura PA317 LXSN 16E6E7 linea di cellule un giorno dopo la semina delle cellule staminali epiteliali primarie con DMEM contenente 10% FBS. (Questa linea produce il LXSN16E6E7 amphotropic retrovirus che codifica per l&#39;HPV16 E6 ed E7 open reading frame, e che può essere utilizzato per infettare in modo stabile e immortalare molti tipi di cellule). </li><li> Trattare PA317 LXSN 16E6E7 linea cellulare con mitomicina C per 2 ore (stesso protocollo con 3T3-J2 cellule). Aggiungi mitomicina C trattati PA317 LXSN 16E6E7 linea cellulare di cellule staminali epiteliali primarie, co-coltura per 6 giorni in KCM con EGF. Rinnova KCM fresco con EGF i media ogni 2 giorni. </li><li> Rimuovere 3T3-J2 e PA317 LXSN 16E6E7 cella con Versene. Aggiungi mitomicina-C trattati 3T3-J2 cellule NHP (neomicina, Hygromycin, puromicina resistente, ~ 200.000 cellule per pozzetto) per le cellule staminali epiteliali. Le celle sono scelti nell&#39;ambito 0,2 mg / ml di G418 (Gibco) per aggiungereitional 6 giorni. Rinnovare fresca KCM G418 contenente con EGF i media ogni 2 giorni. Sopravvivere cellule staminali epiteliali saranno immortalate le cellule staminali. Un bene primario di cellule staminali epiteliali che non sono co-coltura con PA317 LXSN 16E6E7 cellula deve essere impostato come un controllo per la selezione G418, tutte le celle in questo bene dovrebbe essere ucciso da G418 dopo 6 giorni di selezione. </li></ol> 5. Rappresentante Risultati Passaggio iniziale delle cellule staminali epiteliali della pelle e immortalata cellule staminali epiteliali formano colonie stretta costituita da cheratinociti di piccole dimensioni (Figura 2A), circondato da cellule nutrici. Se colta nel siero media liberi con supplementi definito, senza un livello di alimentazione, le cellule staminali si disperderà e non forma colonie stretto (crescono come celle singole e piccoli gruppi). Immortalato le cellule staminali epiteliali mantenere un fenotipo stabile per&gt; 12 mesi di continuo passaggio, ma la tendenza a formare colonie più stretta rispetto alle cellule primarie. Essi non formano colonie di ancoraggio indipendente nei test di agar molle, indicando che queste cellule immortali non possiedono le caratteristiche delle cellule cancerose. Primaria e immortalato le cellule staminali epiteliali esprimere il follicolo pilifero marker delle cellule staminali citocheratina 15 (Figura 3A), e sono in grado di differenziarsi in epidermica, follicolo pilifero e lignaggi sebacee (Figura 3B-D). <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="Figura 1" /> Figura 1. Pizzico capelli. Dopo la spiumatura da dispasi trattati con la pelle, i follicoli dei capelli appaiono come sia capelli telogen club (A) con un ammasso di cellule che circonda la parte inferiore dei capelli, peli o di anagen (B) con un manicotto di epitelio che circonda la lunghezza dei capelli. La regione rigonfiamento telogen forma una regione distinta morfologico di micro-sezionare, mentre il rigonfiamento anagen è meno distinto. Il bulbo contiene anagen cheratinociti della matrice, che rappresenta transito amplificazione cellule che possono essere isolate e coltivate. <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="Figura 2" /> Figura 2. Cellule staminali culture. (A) le cellule staminali epiteliali dalla pelle forma colonie stretto epiteliali (designati da E), quando coltivate in KCM alimentatore con uno strato di cellule (designati da F). (B) espianti follicoli dei capelli dar luogo a escrescenze epiteliali. La regione rigonfiamento telogen (designato dal T) è attaccato al piatto e circondato colonia delle cellule epiteliali insieme con le cellule di alimentazione. <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="Figura 3" /> Figura 3. Differenziazione delle cellule staminali. (A) Le cellule staminali colonie mantenere espressione di marcatori epiteliali come citocheratina 15. (B) le cellule staminali epiteliali della pelle possono essere differenziati lungo il lignaggio follicolo pilifero come determinato dalla espressione K6Hf. (C) Le cellule possono essere coltivate in de-epidermalized derma o altre matrici all&#39;interfaccia aria-liquido e formare una strato di epidermide stratificata con cornified, strato granuloso e strato spinoso. (D) Le cellule staminali possono essere indotte lungo il lignaggio sebacee con olio di globuli rossi positivo.

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Nessun conflitto di interessi dichiarati.

I metodi di estrazione delle cellule e la cultura descritti sono sorprendentemente facili e riproducibili. Abbiamo generato epiteliali colture di cellule staminali da decine di individui in una vasta gamma d&#39;età, inclusi i pazienti con difetti della pelle ereditaria 18. E &#39;meglio iniziare il processo il giorno della raccolta dei tessuti, tuttavia cellule rimangono vitali nel multimediale su ghiaccio per diversi giorni, facilitando la spedizione notte, se necessario. Pelle lifting scartato rendimenti ...

Mezzi di cheratinociti (KCM) [DMEM e Prosciutto s F12 (GIBCO, 3:1), adenina (Sigma, 180 mm), 10% di siero fetale bovino (GIBCO), tossina colerica (ICN, 0.1 nM), penicillina / streptomicina (GIBCO, 100 U / ml e 100 mg / ml, rispettivamente), idrocortisone (Sigma, 0,4 mg / ml, 1,1 mm), T/T3 (transferrina, GIBCO, 5 mg / ml, 649 nm e triiodo-l-thyronine, Sigma, 2 nM) , l&#39;insulina (Sigma, 5 mg / ml, 862 nM) e EGF (Sigma, 10 ng / ml, 1.6 nM), pH 7,2]

isolation and culture of adult epithelial stem cells from human skin

L&#39;omeostasi di tutti i tessuti e auto-rigenerante dipende da cellule staminali adulte. Mentre le cellule staminali indifferenziate subire divisioni asimmetriche, che generano cellule figlie che mantengono il fenotipo delle cellule staminali e di transito-amplificazione cellule (cellule TA) che migrano dalla nicchia delle cellule staminali, subiscono rapida proliferazione e differenziazione terminale per ripopolare il tessuto. Le cellule staminali epiteliali sono state identificate nell&#39;epidermide, follicolo pilifero, e intestino come cellule con un alto potenziale proliferativo in vitro e come slow-ciclismo-label mantenendo le cellule in vivo 1-3. Adulti, tessuto-specifiche cellule staminali sono responsabili della rigenerazione dei tessuti in cui risiedono durante il normale turnover fisiologico, così come durante periodi di stress 4-5. Inoltre, le cellule staminali sono generalmente considerati più potenti, che hanno la capacità di dare origine a più tipi di cellule all&#39;interno del tessuto 6. Per esempio, roditori cellule staminali dei follicoli dei capelli in grado di generare epidermide, ghiandole sebacee e follicoli piliferi 7-9. Abbiamo dimostrato che le cellule staminali dall&#39;umano regione rigonfiamento follicolo pilifero mostra multi-potenzialità 10. Le cellule staminali sono diventate uno strumento prezioso nella ricerca biomedica, per la loro utilità come un sistema in vitro per lo studio della biologia dello sviluppo, la differenziazione, tumorigenesi e per la loro possibile utilità terapeutica. E &#39;probabile che le cellule staminali epiteliali adulte sarà utile nel trattamento di malattie come la displasia ectodermica, monilethrix, Netherton sindrome, malattia di Menkes, epidermolisi bollosa ereditaria e alopecia 11-13. Inoltre, altri problemi della pelle come ad esempio ustioni, ferite e le ulcere croniche potranno beneficiare di terapie con cellule staminali legate 14,15. Dato il potenziale per la riprogrammazione delle cellule adulte in uno stato pluripotenti (cellule iPS) 16,17, il facilmente accessibile ed espandibile cellule staminali adulte di pelle umana può fornire una preziosa fonte di cellule per la terapia di induzione e a valle per una vasta gamma di malattie tra cui diabete e morbo di Parkinson.

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Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Un modo rapido, robusto di isolare vitali cellule staminali epiteliali adulte di pelle umana è descritta. Il metodo utilizza la digestione enzimatica della matrice di collagene della pelle, seguita da spiumatura dei follicoli piliferi e l&#39;isolamento delle sospensioni singola cella o frammenti di tessuto per la coltura cellulare.

Isolamento e la coltura di cellule staminali epiteliali da pelle umana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

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28.2K Views.  University of Massachusetts Medical School. Un modo rapido, robusto di isolare vitali cellule staminali epiteliali adulte di pelle umana è descritta. Il metodo utilizza la digestione enzimatica della matrice di collagene della pelle, seguita da spiumatura dei follicoli piliferi e l'isolamento delle sospensioni singola cella o frammenti di tessuto per la coltura cellulare.

Video: Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Isolation and Large Scale Expansion of Adult Human Endothelial Colony Forming Progenitor Cells

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult human peripheral blood within a mean of 12 days. After large scale expansion &gt;1x108 ECFCs can be obtained for further tests. Advantageously by using pHPL the contact of human cells with bovine serum antigens can be excluded. By flow cytometry and immunohistochemistry the isolated cells can be characterized as ECFC and their in vitro functionality to form vascular like structures can be tested in a matrigel assay. Further these cells can be subcutaneously injected in a mouse model to form functional, perfused vessels in vivo. After long term expansion and cryopreservation proliferation, function and genomic stability appear to be preserved. 3,4
This animal-protein free isolation and expansion method is easily applicable to generate a large quantity of ECFCs.

Endothelial colony forming progenitor cells (ECFCs) are a promising tool to study vascular homeostasis and repair.1,2 This paper introduces a novel animal-serum free method for isolation and expansion of ECFC from heparinised adult human peripheral blood with pooled human platelet lysate (pHPL) diminishing the risk of anti-bovine immunisation.

Isolamento e di espansione su vasta scala di umano adulto cellule progenitrici endoteliali che formano colonie

This paper introduces a novel recovery strategy for endothelial colony forming progenitor cells (ECFCs) from heparinized but otherwise unmanipulated adult ...

Ricerca

Biologia

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

Isolamento di cellule staminali da xenotrapianti di cancro pancreatico umano

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

Naïve Adult Stem Cells isolamento da colture primarie di fibroblasti umani

Over the last decade, several adult stem cell  populations have been identified in human skin 1-4.  The isolation of multipotent adult dermal precursors ...

Induction and Analysis of Epithelial to Mesenchymal Transition

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

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

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

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

Manual Isolation of Adipose-derived Stem Cells from Human Lipoaspirates

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

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

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

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

Isolation, Culture, and Transplantation of Muscle Satellite Cells

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

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

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

Isolamento, Cultura e trapianto di cellule muscolari satellite

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

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

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

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

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

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

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolamento e cultura dei cheratinociti primari del mouse dalla pelle dei neonati e adulti

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...