Yazarlar AZALAN kültürlerle ilk yardım için Xiu-Ping Wang teşekkür ederim. Yazarlar (OH MGC, K08-DE022377-02 AHJ, K12-GM081266 için K99-DE022059 ve ODK R01-DE021420) Ulusal Sağlık Enstitüleri burs ve hibe tarafından finanse edilmektedir.

Yetişkin Fare 1. Dissect Alt Hemi-altçeneler <ol><li> Önce bu protokolü gerçekleştirmek için, gerekli kurumsal onay almak ve tüm hayvan bakımı kurallarına uymak emin olun. Standart prosedürler IACUC onaylı kullanılarak hayvan Euthanize. Bu iş için CO 2 boğulma servikal dislokasyon kullanılmıştır. <ol><li> İSTEĞE BAĞLI: Bir jilet kullanarak vücudun geri kalanından ayrı kafa. </li></ol></li><li> Yaka için alt dudak deri çıkarın. </li><li> Bir neşter kullanılarak, gevşek bağlı iki alt ön dişler arasında bir kesi yapmak. Basınç uygulayarak sonra, mandibula iki parçaya bölünecektir. </li><li> Çene ayırmak için çene kondil ve altçenedeki eklem ve çene kas yanında kesim arasındaki neşter kama. </li><li> Sadece kemik kalana kadar tüm kas, tendon ve bağ çıkarın. </li></ol> 2.. Insizör yalıtmak <ol><li> Bir # 15 neşter kullanılarakDikkatlice membranöz bölgenin yakın ucuna en uzak ucundan itibaren bir 45 ° açıyla kemik tıraş. Kenarında kemik parçaları da dahil olmak üzere kalan kemik uzağa almak için neşter ucu kullanın. Yavaş yavaş sadece 3. molar altında başlayan, mandibulanın lingual kortikal plaka uzakta almaya başlar. </li><li> Kesici CL içeren alanına doğru dikkatli çalışıyor, temiz olana kadar devam edin. </li><li> Inferior alveolar sinir desteyi. </li><li> İlk CL proksimal kemik ve dokuları temizlemek için temiz kesim olun. </li><li> Kabaca 2. molar altında neşter ile distale ikinci bir kesim yapmak. Proksimal kesici bölgesi, daha sonra kemik ile yakın kesici medial yüzeyi arasındaki neşter sokulmasıyla dışarı çıkarılmaktadır. Doku gözyaşı değil, böylece bu dikkatli yapılmalıdır. Kesi hareket proksimal-distal. ALTERNATİF ADIM: Yukarıdaki alan boyunca mümkün olduğu kadar kemik çıkarmakncisor. Alan temizlendikten sonra, dikkatlice emaye bölümünün, CL bölgeye yakın işlemek için bir boyut 5 forseps kullanarak tüm INCISOR kaldırın. </li><li> Düşük yapışma plaka içerisinde 4 ° C&#39;de 3-4 saat boyunca 1X PBS içinde% 2 kolajenaz olarak kesilmiş dokuları (alternatif Aşama 2.5 Şekil 3E olarak izole edilmiş CL, ya da tüm ya da kesici) yerleştirin. 1-10 kesici dişler için, 12 oyuklu bir plaka içerisinde oyuk başına 100 ul kullanın. 10 &#39;den daha büyük kesici için, 6-çukurlu düşük yapışkan bir plaka içerisinde oyuk başına 500 ul kullanın. </li></ol> 3.. Microdissect Labial Servikal döngü <ol><li> Soğuk DMEM/F12 ortamı içinde kolajenaz ve yerden CL bölgesini çıkartın. </li><li> Boyutu 5 forseps veya bir insülin şırıngası (1 cc, 28 G ½) kullanılarak CL alt ucunda yavaşça çekin; epitel bozulmadan mesenkimi ayrı düşecek. Bir &quot;kanat-benzeri&quot; bir yapı olarak yanal uzanır CL ve komşu epitel görünür olacaktır. </li><li> KaldırmakHer iki tarafta bir V-şekilli bir kesi ve hemen buz üzerinde soğuk DMEM/F12 &#39;de yer ile bitişik epitelden apikal tomurcuk. </li><li> 1.5 ml mikrofuge&#39;de tüp toplamak, tüm CLS için tekrarlayın. </li><li> , Numuneler ile mikrofuj tüp aşağı Spin ortamları çıkartın, 100 ul hücre dekolmanı çözümü ile değiştirin. </li><li> 37 ° C&#39;de 30 dakika boyunca hücre ayırma çözeltisi içinde inkübe dokuları </li><li> Hafifçe yukarı ve aşağı pipetleme 1.000 ul düşük yapışma pipet kullanarak tek bir hücre süspansiyonu üretmek için dokuları ayırmak. Hücreler, aynı zamanda ayrışma elde etmek için steril bir hücre süzgecinden yerleştirilebilir. </li><li> Doku kültürü plastik veya diğer istenen malzeme üzerinde hemositometre, plaka hücreleri saymak. </li></ol> DESCs 4. İlköğretim Kültür <ol><li> , 6-çukurlu bir plaka içerisinde Plaka hücreler 1-6 x 10 20 n &#39;lik bir konsantrasyonda fare EGF rekombinant proteini ile takviye edilmiş DMEM/F12 oluşur TANIM ortam içinde 4 hücre / ml,g / ml, 25 ng / ml &#39;lik konsantrasyonda, 1X B27 takviyesi, ve% 1 antibiyotik solüsyon (penisilin, 100 U / ml streptomisin, 50 ug / ml) de FGF rekombinant protein. </li><li> % 5 CO2 TANIM eden 1 ml&#39;lik ortam içinde 37 ° C &#39;de bir 6-yuvalı plaka üzerine kaplama hücreler büyür. Hücre yapışması ve koloni oluşumunu sağlamak için kaplama sonrası 5 gün boyunca ilk kültürleri rahatsız etmeyin. </li><li> Birinci ortam değişikliği için, yeni medya yarı hacmine (500 ul) ile eski ortam hacmine (500 ul) yarısı değiştirin. Ortam değiştirirken mikroskop altında koloniler edin. </li><li> Birinci ortam değişiklik yapıldıktan sonra, her 2 günde bir ortam (1-2 mi) değiştirmek için devam etmektedir. Ortam değiştirirken mikroskop altında koloniler edin. </li></ol> 5.. Passage DESCs <ol><li> Aspire medya ve yıkama hücreleri önceden ısıtılmış PBS ile 3x. </li><li> Tuzlu su çözeltisi içinde önceden ısıtılmış% 0.25 tripsin-EDTA ekleyin ve ayırmak için DESCs için 10-15 dakika boyunca 37 ° C&#39;de inkübe edin. </li><li> Tripsin nötralize etmek için DESC medya ekleyin, yavaşça 15 ml konik tüp hücreleri, yer ve toplamak için doku kültürü plaka boyunca pipetle. </li><li> 2 dakika boyunca 800 xg&#39;de hücreleri aşağı spin. </li></ol>

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Yazarlar ifşa etmek Çıkar çatışması var.

Epitel hücreleri 40 yıl önce 21-24 başarıyla üzerinde ilk kültürlü idi, ve daha yakın zamanda, epitel kök hücrelerin 25-27 başarılı izolasyon epitel biyoloji bilgimizi ilerlemiştir. Biz yetişkin fare kesici dişlerinin DESCs, diş biyoloji ve mine oluşumuna dair önemli bilgiler verim potansiyeline sahip kök hücrelerin nispeten ilgili yeterli nüfusa izole etmek için bir protokol rapor. Bu protokol, ilk olarak kıl folikülü 28 epitel kök hücre izolasyonu daha ön...

Omurgalıların eşsiz özelliklerinden biri diş evrimdir. Moleküler yollar ve bu organın ilgili morfolojik uzmanlık gelişim ve evrimsel biyologlar 5 de dahil olmak üzere, çeşitli açılardan incelenmiştir gibi diş, araştırma birçok alanda önemli bir model sistem haline gelmiştir. Daha yakın zamanda, rejeneratif tıp alanında bir model olarak diş kullanarak değerli bilgiler kazanmaya başladı. Özellikle, diş epitel kök hücrelerin keşif önemli bir ilerleme 6-13 olmuştur. Tüm kemirgenler erişkin kök hücre yönetmeliği incelemek için, bu dişlere erişilebilir bir model sistemi yapım olan büyüme kök hücreleri tarafından körüklenen sürekli büyüyen kesicilerin sahip. Genetik soy izleme deneylerinde 8,9,12,14 ardından 1970&#39;lerde 10,11 etiketleme deneyleri, DESCs kesici proksimal bölgesinde bulunan olduğunu göstermiştir. KökHücre labiyali servikal döngü (CL) olarak bilinen, varsayılan niş, dışarı labial yan hareket epitel bölmeye döl ve geçiş yalıtım (TA) hücreleri (Şekil 1) adı verilen bir hücre popülasyonuna katkıda bulunur. Özellikle, DESCs dış mine epiteli (OEE) ve Stellate retikulum (SR) 8,9,14 ikamet ve iç mine epiteli (IEE) sonra hareket hücre döngüsü sınırlı sayıda ilerlerken TA hücrelere yol açar ve distal kesici uzunluğu (Şekil 1) boyunca. Fare dişe ayırt ameloblastlar tek bir günde 15,16 yaklaşık 350 mikron dikkate değer bir oranda kesici dişin boyunca distale hareket devam ediyor. Onlar hareket ettikçe, hücrelerin olgun ameloblastlar ve stratum intermedium (SI) hücrelerin içine ayırt. Emaye matris tam kalınlık olarak biriktirilmesi sonra ameloblastlar birçok apoptoz geçirir, ve geriye kalan hücreler boyutu küçültmek ve emaye olgunlaşmasını düzenleme &lt;&gt; 17 sup. Gibi SR gibi Labiyal CL, diğer hücre tiplerinin soy daha az açıktır ve mezenşimin 18 ve lingual CL kök hücrelerin ilişkin veriler sadece ortaya çıkmaya başlıyor. Fare modeli kullanılarak kesici, grup bir dizi genetik yollar ve doğal kök hücre bazlı bir organ yenilenmesine katılan hücre biyolojik süreçleri aydınlatmak için çalıştık. Bununla birlikte, dudak CL 5,000 birincil hücreler zorlu çalışma yapar kesici fare başına olduğu tahmin hücrelerin nispeten küçük bir sayısını ihtiva etmektedir. Bu nedenle, kültür çalışmaları yapılmış ve in vivo olarak elde olmayan deneysel yaklaşımlar için yeni kapılar açmak için in vitro 6,16,19,20 bu hücreleri genişletmek edilmiştir. En son çalışma, bu hücreler, kendini yenileme ve kültürlendiğinde 13 hücreleri ifade Amelogenin içine ayırt hem göstermiştir. Burada, th için bir yöntem tarif etmekte ve açıklamaktadırfare labiyali CL hücrelerin e güvenilir ve tutarlı bir kültürü.

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

Diş yenilenme ve yenileme altında yatan hücresel ve moleküler mekanizmaların anlaşılması büyük ilgi 1-4 bir konu haline, ve fare kesici, bu işlemler için bir model sağlar sahiptir. Bu olağanüstü organ hayvanın hayatı boyunca sürekli büyür ve (dil doğru) (dudağa doğru) labial ve lingual servikal döngü (CL) bölgeleri muhafaza yetişkin kök hücrelerin aktif havuzlarından gerekli tüm hücre tiplerini oluşturur. Labiyali CL sadece diş kök hücreler labiyali yüzeyinde emaye, dişlerin dış kaplama oluşturmak ameloblastlar sebebiyet verir. Bu asimetrik mine teşekkülü kesici ucunda aşınma sağlar ve proksimal dişe atalarıdır ve kök hücreler diş dokuları sürekli doldurulan emin olun. Bu in vitro progenitör veya kök hücreleri izole ve büyümek için yeteneğini kendi genişleme sağlar ve saat gibi in vivo elde değil çok sayıda deney, kapılarını açıyorpotansiyel kök hücre düzenleyici faktör üksek throughput testi. Burada, biz tarif ve fare kesici dişin labial CL kültür hücreleri güvenilir ve tutarlı bir yöntem ortaya koymaktadır.

Fare hemi-mandibul biri sürekli büyüyen kesici ve üç köklü azı dişini (Şekil 1A) oluşmaktadır. Tüm diş dentin ve emaye, diş taç iki mineralize bileşenleri (Şekiller 1A ve 1 A &#39;) oluşmaktadır. Incisor evler iki kök hücre nişleri labial CL ve lingual CL denir ve emaye labial tarafında (Şekil 1A &#39;) münhasıran oluşturulmuştur. DESCs kesici emaye oluşumundan sorumlu olan ve labial CL, özellikle OEE ve SR (Şekil 1A&#39;&#39;)</s...

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

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

Sürekli büyüyen fare kesici diş epitel kök hücreler (DESCs) diş dokularının yenilenmesini eğitim için bir model sağlar. Sürekli ve güvenilir bir şekilde kesici bu hücrelerin elde edilmesi ve in vitro ile genişletilmesi için sağlam bir sistem burada bildirilmektedir.

Yetişkin Fare insizör Diş Epitel Kök Hücreleri İzolasyon ve Kültür

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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

Research

JoVE Journal

Biology

15.0K Görüntüleme.  University of California, San Francisco.  Sürekli büyüyen fare kesici diş epitel kök hücreler (DESCs) diş dokularının yenilenmesini eğitim için bir model sağlar. Sürekli ve güvenilir bir şekilde kesici bu hücrelerin elde edilmesi ve in vitro ile genişletilmesi için sağlam bir sistem burada bildirilmektedir. 

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

Isolation of Retinal Stem Cells from the Mouse Eye

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

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

Fare Göz Retina Kök Hücre İzolasyonu

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

Biyoloji

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

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

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

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 ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

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

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

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

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

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Yetişkin İskelet Kası gelen bireysel Myofibers ve Uydu Hücre İzolasyonu ve Kültür

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Na&iuml;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

Over the last decade, several adult stem cell populations have been identified in human skin 1-4. The isolation of multipotent adult dermal precursors was first reported by Miller F. D laboratory 5, 6. These early studies described a multipotent precursor cell population from adult mammalian dermis 5. These cells--termed SKPs, for skin-derived precursors-- were isolated and expanded from rodent and human skin and differentiated into both neural and mesodermal progeny, including cell types never found in skin, such as neurons 5. Immunocytochemical studies on cultured SKPs revealed that cells expressed vimentin and nestin, an intermediate filament protein expressed in neural and skeletal muscle precursors, in addition to fibronectin and multipotent stem cell markers 6. Until now, the adult stem cells population SKPs have been isolated from freshly collected mammalian skin biopsies.
Recently, we have established and reported that a population of skin derived precursor cells could remain present in primary fibroblast cultures established from skin biopsies 7. The assumption that a few somatic stem cells might reside in primary fibroblast cultures at early population doublings was based upon the following observations: (1) SKPs and primary fibroblast cultures are derived from the dermis, and therefore a small number of SKP cells could remain present in primary dermal fibroblast cultures and (2) primary fibroblast cultures grown from frozen aliquots that have been subjected to unfavorable temperature during storage or transfer contained a small number of cells that remained viable 7. These rare cells were able to expand and could be passaged several times. This observation suggested that a small number of cells with high proliferation potency and resistance to stress were present in human fibroblast cultures 7.
We took advantage of these findings to establish a protocol for rapid isolation of adult stem cells from primary fibroblast cultures that are readily available from tissue banks around the world (Figure 1). This method has important significance as it allows the isolation of precursor cells when skin samples are not accessible while fibroblast cultures may be available from tissue banks, thus, opening new opportunities to dissect the molecular mechanisms underlying rare genetic diseases as well as modeling diseases in a dish.

Na&amp;#239;ve Adult Stem Cells Isolation from Primary Human Fibroblast Cultures

We report a method to isolate na&#239;ve multipotent skin-derived precursor (SKP) cells from primary human fibroblast cultures. We show that these SKPs derived from fibroblast cultures share similar stem cell properties to the ones derived directly from human skin biopsies. These cells express the neural crest marker, nestin, in addition to the multipotent markers such as OCT4 and Nanog.

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 ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Mezenkimal Geçiş indüksiyon ve Epitel Analizi

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

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

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

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

Yalıtım, karakterizasyonu ve yetişkin fare kalp kalp kök hücre farklılaşma

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

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

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

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

Yetişkin Fare Kalbinden Atriyal Miyositler, Ventrikül Miyositleri ve Miyosit Olmayanların Eşzamanlı İzolasyonu ve Kültürü

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

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

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

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

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 ...