Este trabajo está financiado por el NIH / NCI subvención R01CA-118916

1. Extracto de las células epiteliales madre de piel humana <ol><li> Antes de iniciar el procedimiento de aislamiento de las células epiteliales del tallo que se necesita para preparar los medios respectivos y los reactivos (ver tabla 1). </li><li> Piel fresca humano adulto cuero cabelludo de los procedimientos de cirugía estética o biopsia por punción se recoge, a continuación, se incuban en FBS DMEM / 10% / Dispasa (4 mg / mL) durante la noche a 4 ° C. Incubación de 2-4 horas a 37 ° C también es efectiva. Trozos de piel debe tener un ancho máximo de 1 cm para permitir la enzima de penetrar. </li><li> Transferencia de la piel en una placa de Petri esterilizadas, saque cada pelo de la piel, sujetando el tallo del pelo cerca de la superficie de la piel y tirando con firmeza y sin problemas. Seleccione los folículos en la fase de telogen en función de su morfología bajo un microscopio de disección, corte de la región bulto (Figura 1) la transferencia de los folículos en un tubo de 15 ml esterilizados. Folículos anágena también se puede utilizar si se corta en 1 / 3 superior del folículo para obtener folículo piloso &quot;bulto&quot; región (fig. 1B). </li><li> Incubar los fragmentos aislados del folículo en una mezcla de 0,05% de tripsina-EDTA (GIBCO) y Versene (0,53 mM EDTA 4NA, GIBCO) (1:1) durante 15-20 minutos a temperatura ambiente con agitación periódica, añadir 4 ml de DMEM + 10 % de SFB para detener la reacción, la desaceleración durante 5 minutos a 800 rpm. Por otra parte, el folículo explantes fragmento de tejido se pueden colocar en la cultura sin la tripsina digerir para cultivar células de los folículos individuales. </li><li> Desechar el sobrenadante con cuidado (~ 0.2-0.5 mL ahorrar para evitar la pérdida de las células) y vuelva a suspender en 1 ml de KCM (medio de queratinocitos), sin FEAG. </li></ol> 2. La cultura primaria de las células madre epiteliales <ol><li> Antes de que el cultivo de células madre epiteliales, la mitomicina C tratados con células 3T3-J2 (células alimentadoras) se deben preparar de la siguiente manera. 3T3-J2 células se cultivaron en DMEM con FBS al 10% antes del tratamiento. Retire el medio de cultivo de células y lavar dos veces con PBS, el tratamiento de células con 15μg / ml de mitomicina C en DMEM sin ​​suero durante 2 horas a 37 ° C, 5% de CO 2. Retire la mitomicina C que contienen DMEM y lavar dos veces con PBS, las células están listas para usar. (Mitomicina C tratados con células 3T3-J2 también se puede preparar previamente y almacenadas a -80 ° C. Descongelar y semillas de las células de un día antes de cultivo de células madre epiteliales primarias, se incuba a 37 ° C, 5% de CO 2. 150.000-200.000 3T3-J2 células por pocillo de la placa de 6 pocillos se recomienda. </li><li> Semilla del folículo del pelo aislado células madre a partir del paso de 1,5 sobre la mitomicina C tratados con células 3T3-J2 en KCM sin ​​noche a la mañana a 37 del FEAG ° C, 5% de CO 2, cambió a EGF contienen KCM al día siguiente. Las células se cultivan a 37 ° C en una atmósfera húmeda con 5% de CO 2. Todos los queratinocitos son alimentados con KCM contiene FEAG cada 2 días y se cultivan durante 14-20 días. Compruebe celular todos los días bajo el microscopio. Células madre del folículo del pelo se forman colonias (Figura 2A). Explantes del folículo del pelo se forman crecimientos después de 10-14 días (Figura 2B). </li></ol> 3. Las células madre epiteliales paso <ol><li> Lavar las células con PBS una vez. Quitar 3T3-J2 células alimentadoras por Versene (pre-calentado a RT) de tratamiento. Incubar la cultura servida en Versene durante 5 minutos, luego agite suavemente y aspirar de células alimentadoras. </li><li> Lave el cabello de las células madre del folículo, ya sea con o Versene tiempo PBS uno. </li><li> Añadir precalentado (a 37 ° C, crítico) de tripsina-EDTA (2 veces) y se incuba a 37 ° C durante 7 minutos o más si es necesario para que la célula madre de los folículos del pelo se puede desprender. No más de 15 minutos es lo mejor. </li><li> Añadir KCM w / o FEAG para detener la tripsina, suavemente la pipeta hacia arriba y abajo para dispersar las células, la desaceleración en 200 g, 5 minutos. </li><li> Vuelva a suspender las células con KCM sin EGF, Conde y re-placa de mitomicina C tratados con 3T3-J2 capa de células. </li></ol> 4. Inmortalizar las células epiteliales madre Las células primarias, células madre adultas, incluso, llegar a la senectud, después del paso de serie y el mes en la cultura. Inmortalización de las células madre primaria es una forma efectiva para aliviar este problema. <ol><li> Plato principal las células madre epiteliales (~ 350,000 células / pocillo de 6 pocillos) en el paso 1 en la capa de alimentación (3T3-J2) y la cultura durante 2 días. </li><li> Cultura PA317 LXSN 16E6E7 línea celular un día después de la siembra de las células epiteliales primarias madre con DMEM con 10% de SFB. (Esta línea produce los LXSN16E6E7 anfotrópico retrovirus que codifica para el VPH 16 E6 y E7 marcos de lectura abierta, y que puede ser usado para infectar de forma estable e inmortalizar muchos tipos de células). </li><li> Tratar PA317 LXSN 16E6E7 línea celular con mitomicina C durante 2 horas (el mismo protocolo con 3T3-J2 células). Añadir mitomicina C tratados PA317 LXSN 16E6E7 línea celular de las células madre epiteliales primarias, co-cultivo durante 6 días en KCM con EGF. Renovar KCM fresco con EGF los medios de comunicación cada 2 días. </li><li> Quitar 3T3-J2 y PA317 LXSN 16E6E7 celda con Versene. Añadir mitomicina-C tratados 3T3-J2 células NHP (neomicina, higromicina, puromicina resistentes, ~ 200.000 células por pocillo) a las células madre epiteliales. Las células se seleccionan menos de 0,2 mg / ml de G418 (Gibco) para añadiritional 6 días. Renovar fresco KCM contiene G418 con EGF los medios de comunicación cada 2 días. Sobrevivir a las células madre epiteliales se inmortalizó células madre. Un pozo de primaria de las células madre epiteliales que no son co-cultivadas con PA317 LXSN 16E6E7 celular se debe establecer como un control para la selección G418, todas las células de este bien debe ser asesinado por G418 después de 6 días de la selección. </li></ol> 5. Resultados representante Pronta aprobación de las células madre de la piel epiteliales e inmortalizado células madre epiteliales forman colonias apretado que consiste en queratinocitos pequeñas (Figura 2A), rodeada de células alimentadoras. Si se cultivan en medio libre de suero con los suplementos definido, sin una capa de alimentación, las células madre se dispersan y forman colonias no ajustados (que crecen como células individuales y pequeños grupos). Inmortalizado células madre epiteliales mantienen un fenotipo estable durante&gt; 12 meses de paso continuo, pero tienden a formar colonias más estrictas que las células primarias. No forman colonias de anclaje independiente en los ensayos de agar blando, lo que indica que estas células inmortalizadas no poseen las características de las células cancerosas. Primaria y inmortalizado células epiteliales expresan madre del folículo piloso marcador de célula madre citoqueratina 15 (Figura 3), y son capaces de diferenciarse en la epidermis del folículo del pelo, y los linajes sebáceas (Figura 3B-D). <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="Figura 1" /> Figura 1. Desplumados pelos. Después de arrancar desde dispasa tratados con la piel, folículos pilosos aparecer como telogen pelos club (A) con una bola de células que rodean la parte inferior del pelo, o pelos anágena (B) con una manga de epitelio que rodea la longitud del pelo. La región del bulbo telogen forma una región distinta a la morfología micro-disección, mientras que el bulto anágena es menos clara. El bulbo anágena contiene queratinocitos de la matriz, lo que representa el tránsito de amplificación de las células que también pueden ser aisladas y cultivadas. <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="Figura 2" /> Figura 2. Células madre culturas. (A) Las células epiteliales se derivan de la piel forma apretada colonias epiteliales (designado por E) cuando se cultivan en KCM con una capa de alimentación de las células (designado por F). (B) explantes del folículo piloso dar lugar a crecimientos epiteliales. La región del bulbo telogen (designado por T) está conectado a la fuente y rodeada de colonias de células epiteliales con células alimentadoras. <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="Figura 3" /> Figura 3. Diferenciación de las células madre. (A) Las células madre colonias mantener la expresión de marcadores epiteliales, como citoqueratina 15. (B) Las células cutáneas epiteliales madre pueden ser diferenciadas a lo largo del linaje folículo del pelo según lo determinado por la expresión K6Hf. (C) Las células pueden ser cultivadas en de-epidermalized dermis u otras matrices en la interfase aire-líquido y se forma una epidermis estratificada con capa córnea, la capa granular y la capa espinosa. (D) Las células madre pueden ser inducidas a lo largo del linaje sebáceas con aceite positiva glóbulos rojos.

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Los métodos de extracción de células y la cultura se describen son sorprendentemente fácil y reproducible. Hemos generado cultivos de células madre epiteliales de docenas de personas a través de un amplio rango de edades, incluidos los pacientes con defectos de la piel hereditaria 18. Lo mejor es comenzar el proceso en el día de la cosecha de tejidos, sin embargo, las células se mantienen viables en los medios de comunicación en hielo durante varios días, lo que facilita el envío de noche si es nec...

Los medios de comunicación de queratinocitos (KCM) [DMEM y Ham s F12 (GIBCO, 3:1), la adenina (Sigma, de 180 mm), el 10% de suero fetal bovino (GIBCO), la toxina del cólera (ICN, 0,1 nM), a la penicilina / estreptomicina (GIBCO, 100 U / ml y 100 mg / ml, respectivamente), hidrocortisona (Sigma, 0,4 mg / ml, 1,1 mM), T/T3 (transferrina, GIBCO, 5 mg / ml, 649 nM, y triyodo-l tironina, Sigma, 2 nM) , la insulina (Sigma, 5 mg / ml, 862 nm), y EGF (Sigma, 10 ng / ml, 1,6 nM), pH 7.2]

isolation and culture of adult epithelial stem cells from human skin

La homeostasis de los tejidos de auto-renovación depende de las células madre adultas. Como células madre indiferenciadas sufren divisiones asimétricas, que generan las células hijas que mantienen el fenotipo de células madre y la amplificación de tránsito de las células (células TA), que migran desde el nicho de células madre, se someten a la rápida proliferación y diferenciación terminal para repoblar el tejido. Las células epiteliales madre han sido identificados en la epidermis, folículos pilosos, y en el intestino como las células con un alto potencial de proliferación in vitro y como lento en bicicleta etiqueta de retención de las células in vivo 1-3. Adulto, el tejido de células madre específicas son responsables de la regeneración de los tejidos en los que residen durante el recambio fisiológico normal, así como en momentos de estrés 4-5. Por otra parte, las células madre son generalmente considerados como multi-potentes, que poseen la capacidad de dar lugar a múltiples tipos de células dentro del tejido 6. Por ejemplo, los roedores células madre del folículo del pelo puede generar epidermis, glándulas sebáceas y los folículos pilosos 7-9. Hemos demostrado que las células madre de la región de bulto del pelo humano folículo exhibición multi-potencialidad 10. Las células madre se han convertido en una herramienta valiosa en la investigación biomédica, debido a su utilidad como un sistema in vitro para el estudio de la biología del desarrollo, la diferenciación, la tumorogénesis y por su posible utilidad terapéutica. Es probable que las células madre adultas del epitelio será útil en el tratamiento de enfermedades como la displasia ectodérmica, monilethrix, el síndrome de Netherton, la enfermedad de Menkes, la epidermólisis bullosa hereditaria y alopecias 11-13. Además, otros problemas de piel como quemaduras, heridas crónicas y úlceras se beneficiarán de las terapias con células madre 14,15. Dado el potencial para la reprogramación de células adultas en un estado pluripotente (células iPS) 16,17, las células adultas de fácil acceso y ampliable madre en la piel humana puede proporcionar una valiosa fuente de células para la inducción y la terapia posterior para una amplia gama de enfermedades, incluyendo la diabetes y la enfermedad de Parkinson.

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Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Una forma rápida y robusta de aislar células viables de adultos epiteliales madre de la piel humana se describe. El método utiliza la digestión enzimática de la matriz de colágeno la piel, seguido por el punteo de los folículos pilosos y el aislamiento de las suspensiones de células individuales o fragmentos de tejido para el cultivo celular.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

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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JoVE Journal

Biology

28.3K vistas.  University of Massachusetts Medical School. Una forma rápida y robusta de aislar células viables de adultos epiteliales madre de la piel humana se describe. El método utiliza la digestión enzimática de la matriz de colágeno la piel, seguido por el punteo de los folículos pilosos y el aislamiento de las suspensiones de células individuales o fragmentos de tejido para el cultivo celular.

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.

El aislamiento de células madre del folículo piloso y queratinocitos epidérmicos de la piel dorsal de ratón

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 ...

Investigación

Biología del Desarrollo

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.

Aislamiento y cultivo de células troncales de la cresta neural de los folículos del cabello humano

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

Neurociencias

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.

Generación y cultivo de Keratinocytes humanos primarios de piel adulto

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 método simplificado y eficiente para aislar Keratinocytes humanos primarios de tejido adulto de la piel

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.

Establecimiento de un sistema de cultivo de esferoides epidérmicos de alto rendimiento para modelar la plasticidad de las células madre de queratinocitos

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.

Aislamiento de fibroblastos dérmicos humanos adultos de la piel abdominal y generación de células madre pluripotentes inducidas utilizando un método no integrador

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

Bioingeniería

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 Aislamiento de Primaria cultivos de fibroblastos humanos

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

Biología

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.

Aislamiento, cultivo y caracterización de células primarias de Schwann, queratinocitos y fibroblastos del prepucio humano

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.

Aislamiento y cultivo de queratinocitos de ratón primario de piel de ratón neonatal y adulto

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.

Aislamiento y cultivo de queratinocitos orales primarios del paladar del ratón adulto

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