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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28.3K 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

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by distinct pools of SCs, which are located in the permanent portion of the HF, a region known as the bulge. These multipotent bulge SCs were initially identified as slow cycling label retaining cells; however, their isolation has been made feasible after identification of specific cell markers, such as CD34 and keratin 15 (K15). Here, we describe a robust method for isolating bulge SCs and epidermal keratinocytes from mouse HFs utilizing fluorescence activated cell-sorting (FACS) technology. Isolated hair follicle SCs (HFSCs) can be utilized in various in vivo grafting models and are a valuable in vitro model for studying the mechanisms that govern multipotency, quiescence and activation.

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS analysis.

Isolare le cellule staminali del follicolo pilifero e epidermico cheratinociti da pelle dorsale del mouse

The hair follicle (HF) is an ideal system for studying the biology and regulation of adult stem cells (SCs). This dynamic mini organ is replenished by ...

Ricerca

Biologia dello sviluppo

Isolation and Culture of Neural Crest Stem Cells from Human Hair Follicles

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a well-characterized niche for adult stem cells1. This segment of the outer root sheath contains a number of different types of stem cells, including epithelial stem cells2, melanocyte stem cells3 and neural crest like stem cells4-7. Hair follicles represent an accessible and rich source for different types of human stem cells. We and others have isolated neural crest stem cells (NCSCs) from human fetal and adult hair follicles4,5. These human stem cells are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They express immature neural crest cell markers but not differentiation markers. Our expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. Differentiated cells not only acquire lineage-specific markers but also demonstrate appropriate functions in ex vivo conditions. In addition, these NCSCs show differentiation potential toward mesenchymal lineages. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. It has been shown that hair follicle derived NCSCs can help nerve regrowth, and they improve motor function in mice transplanted with these stem cells following transecting spinal cord injury8. Furthermore, peripheral nerves have been repaired with stem cell grafts9, and implantation of skin-derived precursor cells adjacent to crushed sciatic nerves has resulted in remyelination10. Therefore, the hair follicle/skin derived NCSCs have already shown promising results for regenerative therapy in preclinical models.
Somatic cell reprogramming to induced pluripotent stem (iPS) cells has shown enormous potential for regenerative medicine. However, there are still many issues with iPS cells, particularly the long term effect of oncogene/virus integration and potential tumorigenicity of pluripotent stem cells have not been adequately addressed. There are still many hurdles to be overcome before iPS cells can be used for regenerative medicine. Whereas the adult stem cells are known to be safe and they have been used clinically for many years, such as bone marrow transplant. Many patients have already benefited from the treatment. Autologous adult stem cells are still preferred cells for transplantation. Therefore, the readily accessible and expandable adult stem cells in human skin/hair follicles are a valuable source for regenerative medicine.

This article presents a robust protocol for isolation and culture of neural crest stem cells from human hair follicles.

L&#39;isolamento e la coltura di cellule staminali neurali Crest di follicoli dei capelli umani

Hair follicles undergo lifelong growth and hair cycle is a well-controlled process involving stem cell proliferation and quiescence. Hair bulge is a ...

Neuroscienze

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

Generazione e la coltura dei Keratinocytes umani primari da pelle adulta

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

A Simplified and Efficient Method to Isolate Primary Human Keratinocytes from Adult Skin Tissue

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical applications. The conventional isolation method of human keratinocytes involves a two-step sequential enzymatic digestion procedure, which has been proven to be inefficient in generating primary cells from adult tissues due to the low cell recovery rate and reduced cell viability. We recently reported an advanced method to isolate human primary epidermal progenitor cells from skin tissues that utilizes the Rho kinase inhibitor Y-27632 in the medium. Compared with the traditional protocol, this new method is simpler, easier, and less time-consuming, and increases epithelial stem cell yield and enhances their stem cell characteristics. Moreover, the new methodology does not require the separation of the epidermis from the dermis, and, therefore, is suitable for isolating cells from different types of adult tissues. This new isolation method overcomes the major shortcomings of conventional methods and is more suitable for producing large numbers of epidermal cells with high potency both for laboratory and for clinical applications. Here, we describe the new method in detail.

Here we present a protocol to efficiently isolate primary human keratinocytes from adult skin tissues. This method simplifies the conventional procedure by using the ROCK Inhibitor Y-27632 in the inoculation medium to spontaneously separate epidermal cells from dermal cells.

Un metodo semplificato ed efficiente per isolare i Keratinocytes umani primari dal tessuto pelle adulta

Primary human keratinocytes isolated from fresh skin tissues and their expansion in vitro have been widely used for laboratory research and for clinical ...

Establishing a High Throughput Epidermal Spheroid Culture System to Model Keratinocyte Stem Cell Plasticity

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human cancers. As a lining tissue, the skin epithelium is often subject to injury and has evolutionarily adapted by acquiring the cellular plasticity necessary to repair damaged tissue. Over the years, several efforts have been made to study epithelial plasticity using in&#160;vitro and ex vivo cell-based models. However, these efforts have been limited in their capacity to recapitulate the various phases of epithelial cell plasticity. We describe here a protocol for generating 3D epidermal spheroids and epidermal spheroid-derived cells from primary neonatal human keratinocytes. This protocol outlines the capacity of epidermal spheroid cultures to functionally model distinct stages of keratinocyte generative plasticity and demonstrates that epidermal spheroid re-plating can enrich heterogenous normal human keratinocytes (NHKc) cultures for integrin&#945;6hi/EGFRlo keratinocyte subpopulations with enhanced stem-like characteristics. Our report describes the development and maintenance of a high throughput system for the study of skin keratinocyte plasticity and epidermal regeneration.

Here we describe a protocol for the systematic cultivation of epidermal spheroids in 3D suspension culture. This protocol has wide-ranging applications for use in a variety of epithelial tissue types and for the modeling of several human diseases and conditions.

Creazione di un sistema di coltura sferoide epidermica ad alto rendimento per modellare la plasticità delle cellule staminali dei cheratinociti

Epithelial dysregulation is a node for a variety of human conditions and ailments, including chronic wounding, inflammation, and over 80% of all human ...

Isolation of Adult Human Dermal Fibroblasts from Abdominal Skin and Generation of Induced Pluripotent Stem Cells Using a Non-Integrating Method

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable diseases, for the reconstitution and regeneration of injured tissues and for the development of new drugs. Despite all the advantages related to the use of iPSCs, such as the low risk of rejection, the lessened ethical issues, and the possibility to obtain them from both young and old patients without any difference in their reprogramming potential, problems to overcome are still numerous. In fact, cell reprogramming conducted with viral and integrating viruses can cause infections and the introduction of required genes can induce a genomic instability of the recipient cell, impairing their use in clinic. In particular, there are many concerns about the use of c-Myc gene, well-known from several studies for its mutation-inducing activity. Fibroblasts have emerged as the suitable cell population for cellular reprogramming as they are easy to isolate and culture and are harvested by a minimally invasive skin punch biopsy. The protocol described here provides a detailed step-by-step description of the whole procedure, from sample processing to obtain cell cultures, choice of reagents and supplies, cleaning and preparation, to cell reprogramming by the means of a commercial non-modified RNAs (NM-RNAs)-based reprogramming kit. The chosen reprogramming kit allows an effective reprogramming of human dermal fibroblast to iPSCs and small colonies can be seen as early as 24 h after the first transfection, even with modifications with the respect to the standard datasheet. The reprogramming procedure used in this protocol offers the advantage of a safe reprogramming, without the risk of infections caused by viral vector-based methods, reduces the cellular defense mechanisms, and allows the generation of xeno-free iPSCs, all critical features that are mandatory for further clinical applications.

Cell reprogramming requires the introduction of key genes, which regulate and maintain the pluripotent cell state. The protocol described enables the formation of induced pluripotent stem cells (iPSCs) colonies from human dermal fibroblasts without viral/integrating methods but using non-modified RNAs (NM-RNAs) combined with immune evasion factors reducing cellular defense mechanisms.

Isolamento di fibroblasti dermici umani adulti dalla pelle addominale e generazione di cellule staminali pluripotenti indotte utilizzando un metodo non integrato

Induced pluripotent stem cells (iPSCs) could be considered, to date, a promising source of pluripotent cells for the management of currently untreatable ...

Bioingegneria

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&#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 ...

Biologia

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

Isolamento, coltura e caratterizzazione di cellule primarie di Schwann, cheratinociti e fibroblasti dal prepuzio umano

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

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 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 ...