Gli autori ringraziano Xiu-Ping Wang aiuto iniziale con culture DESC. Gli autori sono finanziati in parte da borse di studio e sovvenzioni dal National Institutes of Health (K99-DE022059 a AHJ, K12-GM081266 a MGC, K08-DE022377-02 a OH e R01-DE021420 a ODK).

1. Sezionare inferiori Hemi-mandibole di topo adulto <ol><li> Prima di eseguire questo protocollo, avere necessaria l&#39;approvazione istituzionale ed essere sicuri di rispettare tutte le linee guida per la cura degli animali. Euthanize animale utilizzando lo standard IACUC procedure approvate. Per questo lavoro CO 2 asfissia stato usato seguita da dislocazione cervicale. <ol><li> OPTIONAL: testa separata dal resto del corpo utilizzando una lama di rasoio. </li></ol></li><li> Togliere la pelle dal labbro inferiore per la scollatura. </li><li> Usando un bisturi, fare un&#39;incisione tra i due incisivi inferiori, che sono vagamente collegati. Dopo l&#39;applicazione di pressione, la mandibola sarà diviso in due metà. </li><li> Incuneare il bisturi tra il condilo della mandibola e dell&#39;articolazione mandibolare temporale e tagliare lungo il massetere per staccare la mascella. </li><li> Rimuovere tutti i muscoli, tendini e legamenti fino a che solo l&#39;osso rimane. </li></ol> 2. Isolare la Incisor <ol><li> Usando un bisturi # 15, Radere accuratamente l&#39;osso con un angolo di 45 ° dall&#39;estremità distale all&#39;estremità prossimale della regione membranosa. Utilizzare la punta del bisturi per raccogliere via qualsiasi osso residuo compresi i frammenti ossei sul bordo. Lentamente inizia a raccogliere via alla piastra corticale linguale della mandibola, partendo appena sotto il 3 ° molare. </li><li> Continuare fino a quando l&#39;incisivo è pulito, lavorando con attenzione verso la zona contenente la CL. </li><li> Rimuovere inferiore fascio nervo alveolare. </li><li> Fare un taglio netto prima rimuovere l&#39;osso e tessuti prossimale al CL. </li><li> Fare un secondo taglio distale con il bisturi, più o meno sotto il molare 2 °. La regione degli incisivi prossimale è poi sezionato inserendo il bisturi tra l&#39;osso e la superficie mediale dell&#39;incisivo prossimale. Questo deve essere fatto con attenzione, in modo da non strappare il tessuto. Il movimento incisione è prossimale-distale. PASSO ALTERNATIVA: Rimuovere il più possibile osso lungo la zona sopra il incisor. Una zona è azzerato, rimuovere con attenzione tutta la incisivo utilizzando una dimensione di 5 pinze per gestire la porzione di smalto più vicino alla regione CL. </li><li> Posizionare il tessuto sezionato (sia CL isolato come in Figura 3E, o intero incisivo della Fase alternativa 2.5) in 2% di collagenasi in 1X PBS per 3-4 ore a 4 ° C in una piastra bassa aderenza. Per 1-10 incisivi, utilizzare 100 microlitri per pozzetto in una piastra 12 pozzetti. Per maggiore di 10 incisivi, utilizzare 500 microlitri per pozzetto in una piastra da 6 pozzetti bassa aderenza. </li></ol> 3. Microdissect il Labial cervicale Loop <ol><li> Rimuovere regione CL dal collagenasi e il luogo in mezzi DMEM/F12 freddo. </li><li> Tirare leggermente all&#39;estremità inferiore del CL utilizzando dimensioni 5 pinze o una siringa da insulina (1 cc, 28 G ½); mesenchima cadrà a pezzi, mentre l&#39;epitelio rimane intatto. Il CL e l&#39;epitelio adiacente che si estende lateralmente come una struttura &quot;ad ala&quot; saranno visibili. </li><li> Rimuoveregemma apicale dall&#39;epitelio adiacente facendo un&#39;incisione a V su entrambi i lati, e collocare immediatamente in DMEM/F12 freddo su ghiaccio. </li><li> Ripetere per tutti i CL, raccogliere in 1,5 ml provetta per microcentrifuga. </li><li> Spin down provetta per microcentrifuga con campioni, rimuovere i supporti, sostituirli con 100 microlitri soluzione distacco delle cellule. </li><li> Incubare tessuti in soluzione distacco delle cellule per 30 min a 37 ° C. </li><li> Dissociare i tessuti a produrre una sospensione singola cella pipettando gentilmente su e giù con un 1.000 ml a bassa aderenza punta della pipetta. Le cellule possono anche essere immessi attraverso un colino cellula sterile per ottenere dissociazione. </li><li> Contare le cellule con emocitometro, sulla piastra di coltura tissutale di plastica o altro materiale desiderato. </li></ol> 4. Cultura primaria di desc <ol><li> Cellule piastra in una piastra da 6 pozzetti a 1-6 x 10 4 cellule / ml in mezzi desc, che consiste DMEM/F12 addizionato con il mouse EGF proteina ricombinante ad una concentrazione di 20 ng / ml, FGF proteina ricombinante ad una concentrazione di 25 ng / ml, 1X B27 supplemento, e la soluzione 1% di antibiotici (penicillina, 100 U / ml, streptomicina, 50 pg / ml). </li><li> Crescere cellule piastrate su una piastra da 6 pozzetti a 37 ° C in 5% CO 2 in 1 ml di DECR media. Non disturbare culture iniziali per 5 giorni dopo placcatura per consentire l&#39;adesione cellulare e la formazione di colonie. </li><li> Per la prima modifica di supporto, sostituire la metà del volume (500 ml) di vecchi media con metà del volume (500 ml) di nuovi media. Controllare colonie sotto il microscopio quando si cambia supporto. </li><li> Dopo il primo cambio media, continuerà a cambiare media (1-2 ml) ogni 2 giorni. Controllare colonie sotto il microscopio quando si cambia supporto. </li></ol> 5. Desc Passage <ol><li> Aspirare il terreno e lavare le cellule 3x con pre-riscaldato PBS. </li><li> Aggiungere preriscaldata 0,25% tripsina-EDTA in soluzione salina e incubare a 37 ° C per 10-15 min per le desc da staccare. </li><li> Aggiungi mezzi DESC per neutralizzare la tripsina, pipettare delicatamente lungo il tessuto piastra di coltura per raccogliere le cellule, e posto in una provetta da 15 ml. </li><li> Spin le cellule a 800 xg per 2 minuti. </li></ol>

<ol>
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Le cellule epiteliali erano di prima con successo coltivate oltre 40 anni fa 21-24 e, più recentemente, di successo isolamento di cellule staminali epiteliali 25-27 ha avanzato la nostra conoscenza della biologia epiteliale. Riportiamo un protocollo per isolare la desc dell&#39;incisivo topo adulto, una popolazione relativamente poco studiato delle cellule staminali che ha il potenziale per produrre importanti intuizioni in biologia dentale e la formazione dello smalto. Questo protocollo è stato ...

Una delle caratteristiche uniche di vertebrati è l&#39;evoluzione dei denti. Il dente è diventato un importante sistema modello per molte aree di ricerca, come le vie molecolari e specializzazioni morfologiche relativi a questo organo sono stati indagati da diverse prospettive, anche biologi dello sviluppo ed evolutivi 5. Più recentemente, il campo della medicina rigenerativa ha iniziato a ottenere preziose informazioni utilizzando il dente come modello. In particolare, la scoperta delle cellule staminali epiteliali Dental è un importante progresso 6-13. Tutti i roditori possiedono incisivi in ​​continua crescita la cui crescita è alimentata dalle cellule staminali, rendendo questi denti di un sistema modello accessibile a studiare regolamento sulle cellule staminali adulte. Esperimenti di etichettatura nel 1970 10,11, seguita da lignaggio genetico esperimenti di tracciamento 8,9,12,14, hanno dimostrato che desc risiedono nella regione prossimale del incisivo. Lo steloprogenie di cellule nel compartimento epiteliale sul lato labiale mossa dalla nicchia putativo, noto come il ciclo cervicale labiale (CL), e contribuire ad una popolazione di cellule chiamate transito amplificando (TA) cellule (Figura 1). In particolare, desc risiedono nell&#39;epitelio esterno dello smalto (OEE) e stellate reticolo (SR) 8,9,14, e lo smalto epitelio interno (IEE) dà origine alle cellule TA che il progresso attraverso un numero limitato di cicli cellulari e poi mossa distalmente lungo la lunghezza della incisivo (Figura 1). Le ameloblasts differenziazione nel incisivo topo continuano a muoversi distalmente lungo l&#39;incisivo ad un tasso notevole di circa 350 micron in un solo giorno 15,16. Come si muovono, le cellule si differenziano in ameloblasts maturi e strato e transizione (SI) cellule. Dopo aver depositato l&#39;intero spessore della matrice dello smalto, molti dei ameloblasts subiscono apoptosi, e le cellule rimanenti ridursi in dimensione e regolare smalto maturazione &lt;sup&gt; 17. La stirpe degli altri tipi di cellule del labiale CL, come la SR, è meno chiaro, e dati relativi alle cellule staminali nel mesenchima 18 e nella linguale CL stanno solo iniziando a emergere. Utilizzando il modello incisivo del mouse, un certo numero di gruppi hanno lavorato per chiarire i percorsi genetici e cellulari processi biologici coinvolti nella naturale a base di cellule staminali rinnovo dell&#39;organo. Tuttavia, il labiale CL contiene un numero relativamente piccolo di cellule, stimato in circa 5.000 a incisivo mouse, che rende il lavoro con cellule primarie impegnativo. Pertanto, gli sforzi sono stati fatti per la cultura ed espandere queste cellule in vitro 6,16,19,20 per aprire nuove porte per approcci sperimentali che non sono realizzabili in vivo. Lo studio più recente ha dimostrato che queste cellule possono sia auto-rinnovarsi e di differenziarsi in cellule che esprimono amelogenin quando coltivate 13. Qui, descriviamo e dimostrare un metodo per ªe cultura affidabile e coerente delle cellule dal labiale del mouse CL.

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isolation and culture of dental epithelial stem cells from the adult mouse incisor

La comprensione dei meccanismi cellulari e molecolari che sono alla base di rigenerazione del dente e di rinnovamento è diventato un argomento di grande interesse 1-4, e l&#39;incisivo del mouse fornisce un modello per questi processi. Questo notevole organo cresce continuamente per tutta la vita dell&#39;animale e genera tutti i tipi di cellule necessarie dal pool attivi di cellule staminali adulte ospitate nella labiale (verso il labbro) e linguale (verso la lingua) ciclo cervicale (CL) le regioni. Solo le cellule staminali dentarie dal labiale CL danno luogo a ameloblasts che generano smalto, il rivestimento esterno dei denti, sulla superficie labiale. Questa formazione smalto asimmetrico permette all&#39;abrasione sulla punta degli incisivi, e progenitori e cellule staminali nel incisivo prossimale garantire che i tessuti dentali sono costantemente rifornite. La capacità di isolare e coltivare cellule progenitrici o staminali in vitro permette loro espansione e apre porte a numerosi esperimenti non realizzabili in vivo, come high velocità sperimentazione delle potenzialità delle cellule staminali fattori regolatori. Qui, descriviamo e dimostrare un metodo affidabile e coerente per le cellule di coltura dalla CL labiale dell&#39;incisivo mouse.

Il mouse emi-mandibola comprendere una continua crescita incisivo e tre molari radicate (Figura 1A). Tutti i denti sono costituiti da smalto e dentina, i due componenti mineralizzate della corona del dente (Figure 1A e 1A &#39;). Le case incisivi due nicchie staminali chiamato il labiale e linguale CL CL, e smalto è formato esclusivamente sul lato labiale (Figura 1A &#39;). Desc sono responsabili della formazione incisivo smalto e sono alloggiati nella...

Watch this Scientific Journal Video about Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor at JoVE.com

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

Il mouse in continua crescita incisivo fornisce un modello per lo studio rinnovamento dei tessuti dentali da cellule staminali epiteliali dentali (desc). Un sistema robusto per modo coerente e affidabile l&#39;ottenimento di tali cellule dal incisivo e in espansione in vitro è riportato qui.

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-culture-dental-epithelial-stem-cells-from-adult-mouse

Research

JoVE Journal

Biology

15.1K Views.  University of California, San Francisco.  Il mouse in continua crescita incisivo fornisce un modello per lo studio rinnovamento dei tessuti dentali da cellule staminali epiteliali dentali (desc). Un sistema robusto per modo coerente e affidabile l'ottenimento di tali cellule dal incisivo e in espansione in vitro è riportato qui. 

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

Isolation of Retinal Stem Cells from the Mouse Eye

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

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

Isolamento di cellule staminali della retina dalla Eye mouse

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

Ricerca

Biologia

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Generazione di topi derivati ​​da cellule staminali pluripotenti indotte

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

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 ...

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 ...

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

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

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

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

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

Simultaneous Isolation and Culture of Atrial Myocytes, Ventricular Myocytes, and Non-Myocytes from an Adult Mouse Heart

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. While isolating myocytes from neonatal mouse hearts is relatively straightforward, myocytes from the adult murine heart are preferred. This is because compared to neonatal cells, adult myocytes more accurately recapitulate cell function as it occurs in the adult heart in vivo. However, it is technically difficult to isolate adult mouse cardiac myocytes in the necessary quantities and viability, which contributes to an experimental impasse. Furthermore, published procedures are specific for the isolation of either atrial or ventricular myocytes at the expense of atrial and ventricular non-myocyte cells. Described here is a detailed method for isolating both atrial and ventricular cardiac myocytes, along with atrial and ventricular non-myocytes, simultaneously from a single mouse heart. Also provided are the details for optimal cell-specific culturing methods, which enhance cell viability and function. This protocol aims not only to expedite the process of adult murine cardiac cell isolation, but also to increase the yield and viability of cells for investigations of atrial and ventricular cardiac cells.

A method is described for the simultaneous isolation of myocytes and non-myocytes from both the atria and ventricles of a single adult mouse heart. This protocol results in consistent yields of highly viable cardiac myocytes and non-myocytes and details optimal cell-specific culture conditions for phenotyping and in vitro analysis.

Isolamento simultaneo e cultura di miociti atriali, miociti ventricolari e non miociti da un cuore di topo adulto

The isolation and culturing of cardiac myocytes from mice has been essential for furthering the understanding of cardiac physiology and pathophysiology. ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolamento e coltura di cheratinociti orali primari dal palato del topo adulto

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...