Bu çalışma, Ulusal Sağlık Enstitüsü / NCI hibe R01CA-118.916 tarafından finanse edilmektedir

1. İnsan Cilt Epitel Kök Hücreler Özü <ol><li> Epitel kök hücreleri izole işlemine başlamadan önce ilgili medias ve reaktifler (tablo1) hazırlamak gerekiyor. </li><li> Yüz germe işlemleri veya punch biyopsi taze yetişkin insan saçlı deride toplanır, daha sonra DMEM 4 /% 10 FBS / gecede Dispase (4 mg / ml) ° C de inkübe 2-4 saat süreyle inkübasyon 37 ° C de etkilidir. Cilt adet nüfuz enzim için izin maksimum genişliği 1 cm olmalıdır. </li><li> Steril bir Petri kabı içine Transfer cilt, cilt, cilt yüzeyine yakın saç gövdesine açgözlü ve sağlam bir şekilde ve sorunsuz çekerek her saç çekin. Diseksiyon mikroskobu altında kendi morfolojisine göre telojen aşamada follikül seçin, transfer folikülleri (Şekil 1A), 15 ml steril tüpe şişkinliği bölge kesip. Anajen folikülleri 1 / 3 üst folikül kesme, saç folikülü &quot;çıkıntı&quot; bölge (Şekil 1B) elde etmek için de kullanılabilir. </li><li> % 0.05 tripsin-EDTA (Gibco) ve Versene (0.53 mM EDTA 4NA, Gibco) (1:1) karışımı oda sıcaklığında 15-20 dakika süreyle periyodik sallayarak izole folikül parçaları inkübe ekleyebilir, 4 ml DMEM + 10 % FBS 800 rpm&#39;de 5 dakika süreyle durdurmak tepki aşağı spin. Alternatif olarak, folikül doku parçası eksplantlar tek foliküller kültür hücreleri kültür tripsin sindirmek olmadan yerleştirilmiş olabilir. </li><li> Dikkatle supernatant atın (hücreleri kaybetmemek için kaydetmek ~ 0.2-0.5 ml) ve 1 ml EGF olmadan KCM (keratinosit orta), yeniden askıya. </li></ol> 2. Kültür İlköğretim Epitel Kök Hücreler <ol><li> Önce epitel kök hücreler kültür-J2-3T3 hücreleri (besleyici hücreler) aşağıdaki gibi hazırlanmalıdır C-tedavi mitomisin. J2-3T3 hücreleri tedavi öncesi 10% FBS ile DMEM kültür. Kültür ortamı çıkarın ve hücre PBS ile iki kez yıkamak 37 2 saat süreyle serum olmadan DMEM 15μg / ml mitomisin C ° C,% 5 CO 2 hücre tedavisi . Mitomisin C içeren DMEM çıkarın ve PBS ile iki kez yıkayın, hücreler kullanıma hazır. (Mitomisin C-tedavi-J2-3T3 hücreleri de -80 önceden hazırlanmış ve saklanabilir ° C. çözülme ve tohum hücreleri kültür primer epitelyal kök hücreler bir gün önce, 37 inkübe ° C,% 5 CO 2. 150.000-200.000 de ortalama 6-plaka-J2-3T3 hücreleri tavsiye edilir. </li><li> Mitomisin adım 1.5 Tohum izole saç folikülü kök hücre geceleme EGF 37 olmadan KCM 3T3-J2-hücreleri C-tedavi ertesi gün EGF içeren KCM değiştirildi ° C,% 5 CO 2. Hücreler 37 yetiştirilir ° C% 5 CO 2 içeren bir nemli bir atmosfer . Tüm keratinositler KCM içeren EGF ile beslenen her 2 günde bir ve 14-20 gün için yetiştirilmektedir. Mikroskop altında hücre her gün kontrol edin. Saç folikülü kök hücrelerin koloniler (Şekil 2A) oluşturacaktır. Saç folikülü eksplantlar 10-14 gün sonra (Şekil 2B) çıkıntılar oluşturacak. </li></ol> 3. Passage Epitel Kök Hücreler <ol><li> Hücreler PBS ile bir kez yıkayın. Versene (RT önceden ısıtılmış) tedavisi ile 3T3-J2-besleyici hücreler çıkarın. 5 dakika inkübe kültür Versene de bombeli, sonra yavaşça sallamak ve besleyici hücreler aspire. </li><li> Versene veya PBS bir kez ya da kıl folikülü kök hücreler yıkayın. </li><li> Önceden ısıtılmış ekle (37 ° C, kritik) tripsin-EDTA (2X) ve 37 inkübe ° C 7 dakika veya daha uzun bir süre ayırmak için saç folikülü kök hücre için gerekli. Artık az 15 dakika en iyisidir. </li><li> Tripsin durdurmak için KCM w / o EGF ekle yavaşça 200g aşağı dönmeye hücreleri dağıtmak için 5 dakika aşağı yukarı pipet ve. </li><li> Mitomisin C-tedavi 3T3-J2-hücre tabakası EGF, Say ve yeniden plaka olmadan KCM hücreleri yeniden askıya. </li></ol> 4. Epitel Kök Hücreler ölümsüzleştirin İlköğretim hücreleri, hatta erişkin kök hücreler, seri geçit ve kültür ay sonra yaşlanması ulaşır. Birincil kök hücrelerin ölümsüzleşme Bu sorunu gidermek için etkili bir yoldur. <ol><li> Plate 2 gün boyunca birincil epitel kök hücreleri besleyici tabaka geçit 1 (~ 350.000 hücre / 6 plaka) (3T3-J2) ve kültür. </li><li> Kültür PA317 LXSN 16E6E7 hücre hattı DMEM% 10 FBS içeren primer epitelyal kök hücrelerin ekim sonra bir gün. (Bu çizgi HPV16 E6 ve E7 açık okuma çerçeveleri kodlar amphotropic retrovirüs LXSN16E6E7 üretir ve stabil bir şekilde bulaşabilen ve birçok hücre tipleri ölümsüzleştirmek için kullanılabilir). </li><li> PA317 LXSN 16E6E7 hücre hattı 2 saat boyunca mitomisin C (J2-3T3 hücreleri ile aynı protokolü) ile ödüllendirin. Mitomisin C EGF ile KCM 6 gün PA317 LXSN 16E6E7 hücre birincil epitel kök hücreler hattı, ko-kültür tedavi ekleyin. Her 2 günde bir, taze KCM EGF medya yenileyin. </li><li> 3T3-J2 çıkarın ve PA317 LXSN 16E6E7 Versene hücre. Mitomisin-C tedavi 3T3-J2-NHP hücreleri (neomisin, hygromycin, puromycin dayanıklı, iyi başına ~ 200.000 hücre) epitel kök hücreler. Hücreler eklemek için G418 (Gibco) 0.2 mg / ml altında seçilir6 gün itional. EGF medya her 2 günde bir taze G418 içeren KCM yenileyin. Surviving epitel kök hücreler, kök hücreler ölümsüzleştirdi. G418 seçimi için bir kontrol olarak PA317 LXSN 16E6E7 hücre ile birlikte kültür değildir primer epitelyal kök hücrelerin iyi ayarlanması gerekir, bu iyi seçim 6 gün sonra tüm hücreleri G418 tarafından öldürülmüş olmalıdır. </li></ol> 5. Temsilcisi Sonuçlar Deri epitel kök hücre ve epitel kök hücreleri ölümsüzleştirdi erken geçiş, besleyici hücreler ile çevrili küçük keratinositler (Şekil 2A) oluşan sıkı koloniler oluşturur. Besleyici bir tabaka olmadan, tanımlanan takviyeleri ile serum serbest medya kültürü, kök hücreler sıkı koloniler (bunlar tek hücreleri ve küçük kümeler gibi büyümeye) dağıtmak ve formu değil. Ölümsüzleştirilmiştir epitel kök hücreler sürekli geçiş&gt; 12 ay boyunca istikrarlı bir fenotip korumak, ancak birincil hücreler daha sıkı koloniler oluşturmaya eğilimli. Onlar, yumuşak agar tayinlerde bu ölümsüzleştirdi hücrelerin kanserli hücrelerin özelliklere sahip olmadığını belirten ankraj bağımsız koloniler oluşturmak yok. Birincil ve epitel kök hücreleri ölümsüzleştirdi Hem saç folikülü kök hücre belirteci sitokeratin 15 (Şekil 3A) ifade ve epidermal, saç folikülü ve sebase soyları (Şekil 3B-D) ayırt etmek mümkün. <img src="/files/ftp_upload/2561/2561fig1.jpg" alt="Şekil 1" /> Şekil 1. kıllar çekti. Toplandıktan sonra dispase ile tedavi edilen cilt, saç folikülleri telojen (B) bir kol ile saç uzunluğu çevreleyen epitel hücrelerinin saç alt çevreleyen bir top ile kulüp kıllar (A) ya da anagen saçlar ya görünür. Telogen çıkıntı bölge anajen çıkıntı daha belirgin iken, mikro-incelemek için ayrı bir morfolojik bölge oluşturur. Anagen ampul de izole ve kültür olabilir transit yükseltme hücreleri temsil eden matris keratinositler içerir. <img src="/files/ftp_upload/2561/2561fig2.jpg" alt="Şekil 2" /> Şekil 2. Kök hücre kültürleri. (A), cilt hücreleri (F tarafından belirlenmiş) besleyici bir tabaka ile KCM kültür formu sıkı epitel koloniler (E tarafından belirlenmiş) epitelyal kök hücreleri. (B) Saç folikülü eksplantlar epitelyal çıkıntılar doğuran. Telogen çıkıntı bölge (T tarafından belirlenen) çanak bağlı ve besleyici hücreler birlikte epitel hücre kolonisi çevrilidir. <img src="/files/ftp_upload/2561/2561fig3.jpg" alt="Şekil 3" /> Şekil 3. Kök hücre farklılaşması. (A) Kök hücreler koloniler sitokeratin 15 gibi epitel belirteçleri ifade korumak. (B) Cilt epitel kök hücreler, K6Hf ifade tarafından belirlenen saç folikülü soy boyunca ayırt edilebiliyor. (C), hava-sıvı arayüzü de-epidermalized dermis veya diğer matrisler hücreler kültür ve açığa çıkan tabaka, granüler tabaka ve spinöz tabakası ile tabakalı epidermis oluşturacak. (D) Kök hücreler Oil Red olumlu globülleri yağ soy boyunca teşvik edilebilir.

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Açıklanan hücre çıkarma ve kültür yöntemleri, şaşırtıcı derecede uyduruk ve tekrarlanabilir. Biz, kalıtsal bir cilt kusurları 18 olan hastalar da dahil olmak üzere geniş bir yaş yelpazesinde bireylerin onlarca, epitel kök hücre kültürleri var. Doku hasat gününde işlemine başlamak için en iyisidir, ancak hücreleri gecede nakliye kolaylaştırmak gerekirse, birkaç gün boyunca buz üzerinde medyada canlı kalacaktır. Atılan facelift cilt, tek bir hücre süspansiyonları olarak h?...

Keratinosit medya (KCM) [DMEM ve Ham F12 (Gibco, 3:1), adenin (Sigma, 180 mM),% 10 fetal sığır serumu (Gibco), kolera toksini (ICN, 0.1 nM), penisilin / streptomisin (Gibco, 100 U / ml ve sırasıyla 100 mg / ml), hidrokortizon (Sigma, 0.4 mg / ml, 1.1 mM), T/T3 (transferrin Gibco, 5 mg / ml, 649 nM ve triiodo-l-thyronine, Sigma, 2 nM) , insülin (Sigma, 5 mg / ml, 862 nM) ve EGF (Sigma, 10 ng / ml, 1.6 nM), pH 7,2]

isolation and culture of adult epithelial stem cells from human skin

Kendini sürekli yenileyen dokuların homeostazı yetişkin kök hücreleri üzerinde bağlıdır. Farklılaşmamış kök hücreleri asimetrik bölünmeler tabi olarak, onlar, kök hücre fenotipi ve kök hücre niş göç, hızlı bir şekilde çoğalması geçmesi ve ölümcül doku yeniden doldurmanız ayırt etmek için transit yükseltme hücreleri (TA hücreleri) korumak kızı hücreler üretir. Epidermis, saç folikülü ve bağırsak epitel kök hücreleri, in vitro proliferatif potansiyeli yüksek ve yavaş bisiklet hücreleri in vivo 1-3 etiket istinat hücreler olarak tespit edilmiştir. Yetişkin, doku spesifik kök hücrelerin, normal fizyolojik ciro sırasında yanı sıra stres 4-5 zamanlarda yaşadığınız dokuların rejenerasyonu için sorumludur . Ayrıca, kök hücreler genellikle 6 doku içinde birden fazla hücre tiplerinin ortaya çıkmasına neden kapasitesine sahip, çok güçlü olması için kabul edilir. Örneğin, kemirgen saç folikülü kök hücreler, epidermis, yağ bezleri, ve saç foliküllerinin 7-9 üretebilir . Biz insan saç folikülü çıkıntı bölge kök hücrelerinin çok-potansiyellik 10 sergilediklerini göstermiştir. Kök hücreler, biyomedikal araştırma, gelişim biyolojisi, farklılaşma, tümörogenez ve onların olası tedavi yarar için eğitim in vitro sistemi olarak yarar nedeniyle değerli bir araç haline gelmiştir. Bu, yetişkin epitel kök hücreleri ektodermal displazileri, monilethrix, Netherton sendromu, Menkes hastalığı, kalıtsal epidermolizis bülloza ve alopesilerde 11-13 gibi hastalıkların tedavisinde yararlı olacağı muhtemeldir . Ayrıca, yanık yaraları, kronik yaralar ve ülser gibi diğer cilt problemleri, terapiler 14,15 ile ilgili kök hücre fayda sağlayacaktır . Pluripotent devlet (iPS hücreleri) 16,17 yetişkin hücreleri yeniden programlama için potansiyel göz önüne alındığında, insan derisi kolaylıkla erişilebilir ve genişletilebilir bir yetişkin kök hücre hastalığı da dahil olmak üzere geniş bir yelpazede hücrelerinin indüksiyonu ve alt tedavisi için değerli bir kaynak sağlayabilir diyabet ve Parkinson hastalığı.

Watch this Scientific Journal Video about Isolation and Culture of Adult Epithelial Stem Cells from Human Skin at JoVE.com

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

Insan derisi canlı yetişkin epitelyal kök hücreleri izole bir hızlı, sağlam bir şekilde açıklanmıştır. Bu yöntem cilt kollajen matriks enzimatik sindirim, saç folikülleri ve hücre kültürü için, tek bir hücre süspansiyonları ya da doku parçalarının izolasyonu koparma kullanır.

İnsan Cilt Yetişkin Epitel Kök Hücre İzolasyonu ve Kültür

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

isolation-and-culture-of-adult-epithelial-stem-cells-from-human-skin

Research

JoVE Journal

Biology

28.3K Görüntüleme.  University of Massachusetts Medical School. Insan derisi canlı yetişkin epitelyal kök hücreleri izole bir hızlı, sağlam bir şekilde açıklanmıştır. Bu yöntem cilt kollajen matriks enzimatik sindirim, saç folikülleri ve hücre kültürü için, tek bir hücre süspansiyonları ya da doku parçalarının izolasyonu koparma kullanır.

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.

Dorsal Fare Cilt Saç Folikül Kök Hücreler ve epidermal keratinositler Ayırma

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 ...

Gelişim Biyolojisi

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.

İnsan Saç köklerinin arasından Sinir Crest Kök Hücre İzolasyonu ve Kültür

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

Nörobilim

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.

Oluşturma ve birincil insan lenfositi yetişkin cilt, kültür

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.

Birincil insan Lenfositi yetişkin cilt dokusundan izole etmek için basit ve etkili bir yöntem

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.

Keratinosit Kök Hücre Plastisitesini Modellemek için Yüksek Verimli Epidermal Sferoid Kültür Sistemi Kurulması

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.

Erişkin İnsan Dermal Fibroblastlarının Abdominal Deriden İzolasyon ve İndüklenen Pluripotent Kök Hücrelerin Indüklenen Alacalı Kök Hücrelerin Indükleyici Olmayan Yöntemle Üretilmesi

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

Biyomühendislik

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.

İlköğretim İnsan fibroblast Kültürler gelen naif Erişkin Kök Hücre İzolasyon

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

Biyoloji

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.

İnsan Sünnet Derisinden Primer Schwann Hücrelerinin, Keratinositlerin ve Fibroblastların İzolasyonu, Kültürü ve Karakterizasyonu

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.

Yenidoğan ve Erişkin Fare Ciltlerindeki Primer Fare Keratinositlerin İzolasyonu ve Kültürü

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.

Yetişkin Fare Damak Tadından Birincil Oral Keratinositlerin İzolasyonu ve Kültürü

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