Este trabalho é financiado pelo NIH / NCI conceder R01CA-118916

1. Extrair células-tronco epiteliais da pele humana <ol><li> Antes de iniciar o procedimento de isolar células-tronco epiteliais é preciso preparar os respectivos meios e reagentes (ver tabela 1). </li><li> Frescos adulto pele do couro cabeludo humano de procedimentos facelift ou biópsia com punch é coletado e, em seguida incubar em DMEM FBS / 10% / Dispase (4 mg / mL) durante a noite a 4 ° C. Incubação por 2-4 horas a 37 ° C também é eficaz. Pedaços de pele deve ser uma largura máxima de 1 cm para permitir a enzima para penetrar. </li><li> Transferir a pele em uma placa de Petri esterilizada, retire cada cabelo da pele, segurando o eixo do cabelo perto da superfície da pele e puxando com firmeza e sem sobressaltos. Selecione folículos na fase telógena com base na sua morfologia em um microscópio de dissecação, cortar a região protuberância (Figura 1A) folículos transferência para um tubo de 15 mL esterilizados. Folículos anágenos também pode ser usado se cortado em cima 1 / 3 do folículo para obter folículo piloso &quot;protuberância&quot; região (Figura 1B). </li><li> Incubar os fragmentos folículo isolado em uma mistura de 0,05% de tripsina-EDTA (GIBCO) e Versene (0,53 mM EDTA 4NA, GIBCO) (1:1) para 15-20 minutos em temperatura ambiente com agitação periodicamente, adicionar 4 mL DMEM + 10 FBS% para parar a reação, spin baixo por 5 min a 800 rpm. Alternativamente, explantes folículo fragmento de tecido podem ser colocados em cultura sem tripsina digerir a cultura de células de folículos único. </li><li> Elimine o sobrenadante com cuidado (salvo ~ mL 0,2-0,5 para evitar a perda de células) e re-suspensão em 1 mL KCM (médio queratinócitos), sem EGF. </li></ol> 2. Cultura primária epitelial Células-Tronco <ol><li> Antes de cultivar as células-tronco epiteliais, mitomicina C-tratados 3T3-J2 células (células de alimentação) deve ser preparado da seguinte forma. 3T3-J2 células são cultivadas em DMEM com 10% de FBS antes do tratamento. Remova a mídia de cultura de células e lavar duas vezes com PBS, tratar células com mitomicina C 15μg / mL em DMEM sem soro para 2 hr a 37 ° C, 5% CO 2. Remova a mitomicina C contendo DMEM e lavar duas vezes com PBS, as células estão prontas para uso. (Mitomicina C tratados com células 3T3-J2 também pode ser preparado previamente e armazenadas a -80 ° C. Descongele e semente as células um dia antes de cultivo primário de células-tronco epiteliais, incubar a 37 ° C CO, 5% 2. 150.000-200.000 3T3-J2 células por poço da placa 6-bem é recomendado. </li><li> Sementes isoladas células-tronco do folículo de cabelo a partir do passo 1.5 em mitomicina C-tratados 3T3-J2 células em KCM sem EGF durante a noite a 37 ° C, 5% CO 2, alterado para EGF contendo KCM no dia seguinte. As células são cultivadas a 37 ° C em uma atmosfera úmida contendo 5% de CO 2. Todos os queratinócitos são alimentados com KCM contendo EGF a cada 2 dias e cultivadas por 14-20 dias. Verifique celular todos os dias sob o microscópio. Células-tronco do folículo do cabelo irá formar colônias (Figura 2A). Explantes folículo piloso formarão outgrowths após 10-14 dias (Figura 2B). </li></ol> 3. Células-tronco epiteliais passagem <ol><li> Lavar as células com PBS uma vez. Remover as células 3T3-J2 alimentador por Versene tratamento (pré-aquecido a RT). Incubar a cultura dished em Versene por 5 minutos, em seguida, agite e aspirar as células do alimentador. </li><li> Lave as células-tronco do folículo de cabelo com qualquer Versene ou tempo PBS um. </li><li> Adicionar pré-aquecido (a 37 ° C, crítico) de tripsina-EDTA (2X) e incubar a 37 ° C por 7 minutos ou mais se necessário para a célula-tronco de folículo piloso para separar. Não mais que 15 minutos é o melhor. </li><li> Adicionar KCM w / o EGF para parar a tripsina, gentilmente pipeta cima e para baixo para dispersar as células, spin baixo em 200g, a 5 minutos. </li><li> Re-suspender as células com KCM sem EGF Count, e re-placa em mitomicina C tratados com camada de células 3T3-J2. </li></ol> 4. Imortalizar Células-Tronco Epiteliais Pilhas, células-tronco adultas, mesmo, chegar a senescência após a passagem de série e meses em cultura. Imortalização de células-tronco primário é uma forma eficaz para aliviar este problema. <ol><li> Placa primária de células-tronco epiteliais (~ 350 mil células / poço de 6 de placa bem) em uma passagem sobre a camada de alimentação (3T3-J2) e cultura para 2 dias. </li><li> Cultura PA317 LXSN linha celular 16E6E7 um dia após a semeadura primária de células-tronco epiteliais com DMEM contendo 10% FBS. (Esta linha produz o LXSN16E6E7 retrovírus amphotropic que codifica o HPV16 E6 e E7 quadros de leitura aberta, e que pode ser usado para infectar e se estavelmente imortalizar muitos tipos de células). </li><li> Tratar PA317 LXSN linha celular 16E6E7 com mitomicina C por 2 horas (mesmo protocolo com 3T3-J2 células). Adicionar mitomicina C tratados PA317 LXSN linha celular 16E6E7 ao primário de células-tronco epiteliais, co-cultura por 6 dias no KCM com EGF. Renovar KCM fresco com EGF media a cada 2 dias. </li><li> Remover 3T3-J2 e PA317 LXSN 16E6E7 célula com Versene. Adicionar mitomicina-C trataram células 3T3-J2 NHP (neomicina, higromicina, puromicina resistentes, ~ 200.000 células por poço) para as células-tronco epiteliais. Células estão selecionadas em 0,2 mg / ml de G418 (Gibco) para adicionaritional 6 dias. Renovar KCM G418 fresco contendo com EGF media a cada 2 dias. Sobrevivendo células-tronco epiteliais será imortalizado células-tronco. Um bem de células-tronco epiteliais primários que não são co-cultivados com PA317 LXSN 16E6E7 célula deve ser definido como um controlo para a selecção G418, todas as células desse bem deve ser morto por G418 após 6 dias de seleção. </li></ol> 5. Resultados representante Passagem precoce de células epiteliais da pele-tronco e células-tronco epiteliais imortalizada formam colónias apertado consistindo de pequenas queratinócitos (Figura 2A) rodeado por células alimentadoras. Se cultivadas em soro de mídia livre com suplementos definido, sem uma camada de alimentação, as células-tronco se dispersa e não formam colônias apertado (elas crescem como células únicas e pequenos aglomerados). Células-tronco epiteliais imortalizada manter um fenótipo estável por&gt; 12 meses de passagem contínua, mas tendem a formar colônias mais apertado do que as células primárias. Eles não formam colônias de ancoragem independente em ensaios agar suave, indicando que estas células imortalizadas não possuem as características de células cancerosas. Primário e as células-tronco epiteliais imortalizada expressar folículo piloso marcador de células-tronco citoqueratina 15 (Figura 3A), e são capazes de se diferenciar em folículo piloso epidérmica, e linhagens sebáceas (Figura 3B-D). <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="Figura 1" /> Figura 1. Dedilhados cabelos. Após arrancar a partir de dispase tratados com pele, folículos pilosos aparecem tanto como os cabelos telógeno clube (A) com uma bola de células em torno do fundo do cabelo, ou cabelos anágenos (B) com uma manga de epitélio em torno do comprimento do cabelo. A região bojo telógeno forma uma região distinta morfológicas para micro-dissecção, enquanto o bojo anágena é menos distintas. A lâmpada contém anagen queratinócitos da matriz, o que representa trânsito ampliando-células que também podem ser isoladas e cultivadas. <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="Figura 2" /> Figura células 2. Culturas Stem. (A) células-tronco epiteliais da pele forma apertada colônias epiteliais (designado por E), quando cultivadas em KCM com uma camada de células de alimentação (designados por F). (B) explantes follicle do cabelo dar origem a outgrowths epiteliais. A região bojo telógeno (designada por T) é anexado ao prato e cercada de colônias de células epiteliais com células alimentadoras. <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="Figura 3" /> Figura 3. Diferenciação de células-tronco. (A) As células-tronco manter colônias expressão de marcadores epiteliais como citoqueratina 15. (B) células-tronco epiteliais de pele pode ser diferenciada ao longo da linhagem folículo piloso como determinado pela expressão K6Hf. (C) As células podem ser cultivadas em de-epidermalized derme ou outras matrizes na interface ar-líquido e formar uma camada de epiderme estratificada com cornified, camada granular e camada espinhosa. (D) As células-tronco podem ser induzidas ao longo da linhagem sebáceas com Oil Red glóbulos positivo.

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Os métodos de extração de células e cultura descritos são surpreendentemente fáceis e reprodutíveis. Temos gerado culturas de células-tronco epiteliais de dezenas de indivíduos através de uma faixa etária ampla, incluindo pacientes com defeitos da pele herdado 18. É melhor para começar o processo no dia da colheita de tecidos, no entanto células permanecerá viável nos meios de comunicação sobre o gelo por vários dias, facilitando o transporte de noite, se necessário. Pele facelift descarta...

Media dos queratinócitos (KCM) [DMEM e Ham s F12 (GIBCO, 3:1), adenina (Sigma, 180 mM), 10% de soro fetal bovino (GIBCO), a toxina do cólera (ICN, 0,1 nM), a penicilina / estreptomicina (GIBCO, 100 U / ml e 100 mg / ml, respectivamente), hidrocortisona (Sigma, 0,4 mg / ml, 1,1 mM), T/T3 (transferrina, GIBCO, 5 mg / ml, 649 nM e triiodo-l-thyronine, Sigma, 2 nM) , a 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

A homeostase de todos os tecidos auto-renovação é dependente das células-tronco adultas. Como células-tronco indiferenciadas sofrer divisões assimétricas, que geram células-filhas que mantêm o fenótipo de células-tronco e trânsito ampliando-cells (células TA) que migram do nicho de células-tronco, são submetidos a rápida proliferação e diferenciação terminal para repovoar o tecido. Células-tronco epiteliais foram identificados na epiderme, folículo piloso, e intestino como células com alto potencial proliferativo in vitro e como o ciclismo slow-label de retenção de células in vivo 1-3. Adulto, tecido-específicos de células-tronco são responsáveis ​​pela regeneração dos tecidos em que residem durante turnover fisiológico normal, assim como em épocas de estresse 4-5. Além disso, as células-tronco são geralmente consideradas como multi-potentes, possuindo a capacidade de dar origem a vários tipos de células dentro do tecido 6. Por exemplo, roedor cabelos células-tronco do folículo pode gerar epiderme, glândulas sebáceas e folículos pilosos 7-9. Nós mostramos que as células-tronco do humano região bojo folículo piloso apresentam potencialidade multi-10. Células-tronco tornaram-se uma ferramenta valiosa na pesquisa biomédica, devido à sua utilidade como um sistema in vitro para estudar a biologia do desenvolvimento, diferenciação, tumorigênese e para a sua eventual utilidade terapêutica. É provável que células-tronco adultas do epitélio será útil no tratamento de doenças como displasias ectodérmicas, monilethrix, síndrome de Netherton, doença de Menkes, epidermólise bolhosa hereditária e alopecias 11-13. Além disso, outros problemas de pele, como queimaduras, feridas crônicas e úlceras irá beneficiar de terapias com células-tronco relacionadas 14,15. Dado o potencial para a reprogramação de células adultas em estado pluripotentes (células iPS) 16,17, as células adultas de fácil acesso e expansível-tronco na pele humana pode fornecer uma valiosa fonte de células para a indução e terapia a jusante de uma ampla gama de doenças, incluindo diabetes e doença de Parkinson.

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Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Uma maneira rápida e robusta de isolar células-tronco adultas viável epiteliais da pele humana é descrito. O método utiliza a digestão enzimática da matriz de colágeno da pele, seguido por arrancar dos folículos pilosos e isolamento de células em suspensão simples ou fragmentos de tecido para cultura de células.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele 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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28.3K visualizações.  University of Massachusetts Medical School. Uma maneira rápida e robusta de isolar células-tronco adultas viável epiteliais da pele humana é descrito. O método utiliza a digestão enzimática da matriz de colágeno da pele, seguido por arrancar dos folículos pilosos e isolamento de células em suspensão simples ou fragmentos de tecido para cultura de células.

Vídeo: 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.

Isolando células-tronco do folículo piloso e epidérmico queratinócitos da Dorsal rato Pele

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 ...

Biologia do Desenvolvimento

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.

Isolamento e Cultura de células da crista neural-tronco de folículos capilares humanos

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

Neurociência

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.

Geração e cultivo de queratinócitos humanos primários da pele de 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.

Um método simplificado e eficiente para isolar os queratinócitos humanos primários do tecido da pele de adultos

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.

Estabelecendo um sistema de cultura esferoide epidérmico de alta produtividade para modelar a plasticidade das células-tronco de ceratinócito

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 de fibroblastos dérmicos humanos adultos da pele abdominal e geração de células-tronco pluripotentes induzidas usando um método não integrador

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

Bioengenharia

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 de culturas primárias 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 ...

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, cultura e caracterização de células primárias de schwann, queratinócitos e fibroblastos de Foreskin 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.

Isolamento e Cultura de Queratinócitos de Rato Primário de Pele de Rato Neonatal e 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.

Isolamento e Cultura de Queratinócitos Orais Primários do Paladar do Rato Adulto

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