Bu çalışma (HPL için) NIH hibe R01 GM087609, (MTL) (HPL için) Anthony Shu, NIH hibe U01 HL099776 onuruna Ingrid Lai ve Bill Shu bir Hediyesi, Hagey Laboratuvarı için bir hibe ile kısmen desteklenmiştir Pediatrik Rejeneratif Tıp ve Meşe Vakfı (MTL, GCG ve HPL). GGW Tıp Stanford School, Stanford Tıp Bilim Adamı Yetiştirme Programı tarafından desteklenen ve NIGMS eğitim bursu GM07365 oldu. ZNM Plastik Cerrahi Vakfı Araştırma Bursu Grant ve Hagey Aile Fonu tarafından desteklenmiştir. MSH California Rejeneratif Tıp Enstitüsü (CIRM) Klinik Fellow eğitim bursu TG2-01159, Çene Hastalıkları Cerrahlar American Society (ASMS) / Çene-Yüz Cerrahları Vakfı (MSF) Araştırma Bursu Ödülü ve Transplantasyon ve Doku Mühendisliği Bursu Ödülü tarafından desteklenmiştir.

Bu protokol Laboratuar Hayvanları Bakım Stanford Üniversitesi İdari Heyeti tarafından onaylanan yöntemler izler. Fare Dermisinde 1. Sindirim <ol><li> Ketamin 100 mg / kg + ksilazin 20 mg / kg asepromazin + 3 mg / kg karın içinden enjeksiyonu ile anestezi sonra servikal dislokasyon ile fareler öldürülür. Not: Çeşitli yaş ve arka kullanılabilir. </li><li> Tıraş ve dorsal cilt atmaktadır. Yaklaşık olarak 100,000 hücre sırt derisine bir parça x 100 mm, 60 mm izole edilebilir. </li><li> Kuruması için temiz, steril bir yüzey üzerinde% 70 etanol ve yerinde fareyi Batmak. </li><li> Hemen steril diseksiyon makas kullanarak dorsal fare cilt hasat. Dişi fareler içinde, meme dokusu da dahil olmak üzere kaçının. </li><li> Kuyruk tabanı başlayan çadır kadar cilt için forseps kullanabilir ve üstü fasiyal düzlemi boyunca diseksiyon önce enine kesim yapmak. </li><li> Dikkatle hasat sırasında herhangi deri altı yağ içeren kaçınmakcilt. Herhangi bir deri altı yağ için hasat cildi inceleyin ve dikkatli bir neşter künt kenarını kullanarak kazıyın. </li><li> Buz üzerinde PBS yıkar 5x ardından Betadin hasat cildi durulayın. Not: kirlenmesini önlemek için mümkün olduğunca steril yakın olarak cildi korumak önemlidir. </li><li> Jilet kullanarak ve örnek 2-3 mm&#39;lik parçalar ile düzgün bir tutarlılık kadar steril bir tabak içinde makas diseksiyon cilt kıyma. </li><li> DMEM içindeki 1 mg / ml &#39;lik bir konsantrasyonda, 50 ml, 20 ml kolajenaz IV ihtiva eden konik tüpler hazırlayın. Tüp başına beş farenin bazında tüplere dermis bölün. </li><li> Bir su banyosu ya da fırın ya 1 saat süreyle 37 ° C&#39;de inkübe kuvvetli şekilde örnekleri çalkalayın. </li><li> Inkübatör örnekleri çıkarın ve steril bir muhafaza içinde bir iğne 3-5x olmayan bir 10 ml şırınga geçer. </li><li> 37 ° C&#39;de inkübatör içinde numuneler ve 30 dakika boyunca kuvvetli bir şekilde çalkalanır. </li><li> Steril bir başlık olarakBir 10 ml pipet kullanarak ve 3-5x aşağı örnekleri pipetle. Yeni 50 ml konik tüp içine 100 mikron filtre örneği pipetle. </li><li> Hücre verimi maksimize etmek ve 40 ml toplam hacmi getirmek için, aynı filtre boyunca% 10 FBS DMEM 20 ml geçirin. 4 ° C &#39;de 8 dakika boyunca 300 g&#39;de santrifüjleyin. </li><li> İlk süpernatant kalan öncesinde üst yağ tabakasını kaldırmak için büyük bir özen, bir steril cam pipet kullanarak süpernatantı. Not: Bu adım adiposit kirlenmeyi azaltmak için çok önemlidir. </li><li> 20 ml% 10 FBS DMEM pelet yeniden süspanse edin. </li><li> 70 mikron filtreden geçirilerek hücre / DMEM süspansiyonu geçirin. </li><li> 4 ° C &#39;de 8 dakika boyunca 300 g&#39;de 10 mi,% 10 FBS DMEM ile filtre ve santrifüj filtre süspansiyonu çalkalayınız. </li><li> Yine ilk kalan yağ tabakası kaldırmak için özen, steril bir cam pipet kullanarak süpernatantı. </li><li> Önemli RBC kirlenme varsa (pelet gözle görülür kırmızı), pelet yeniden askıyave 20 ml ACK lizis tamponu içinde, oda sıcaklığında 5 dakika inkübe edilir. Aksi halde 24 adıma atlayın. </li><li> FACS tamponu (PBS,% 10 FBS,% 0.1 sodyum azid) içinde eşit bir hacmi (20 mi) ilave edilir, daha sonra karıştırılır ve bir boyanmamış bir kontrol olarak bir kenara 5 ml&#39;lik bir şişe tutun. 4 ° C &#39;de 8 dakika boyunca 300 g&#39;da kalan santrifüjleyin. </li><li> Süpernatantı ve buz üzerinde pelet koydu. FACS zamanı mevcut değilse, bu noktada hücreler, aşağı dondurulabilir. </li></ol> FACS Fibroblast 2. izolasyonu <ol><li> Her pelet için soy antikor inkübasyon karışımı 500 ul olun. CD45 (1: 200), Tie2 (01:50), Ter: İlk bir tübe DNaz (10 ug / ml) ihtiva eden FACS tampon 475 ul ekleyerek ve daha sonra fluorofor-konjuge CD31 (100 1) ilave edilerek, bu fazlası -119 (1: 200) ve EpCAM (1: 100) antikorları her bir antikor için, ilgili seyreltme elde etmek. </li><li> Nesilli antikor inkübasyon karışımı 500 ul her bir pelet yeniden askıya ve 20 dakika boyunca buz üzerinde, bu süspansiyon inkübe edilir. </li><li> 5 ekleyinörnek DNaz (10 ug / ml) ihtiva eden ve hafifçe karıştırın mi FACS tamponu. 4 ° C &#39;de 8 dakika boyunca 300 g&#39;de santrifüjleyin. </li><li> Süpernatantı ve adım 26&#39;daki gibi 5 ml FACS DNaz ihtiva eden tampon (10 ug / ml) ve aynı koşullar kullanılarak santrifüj ile hücre pelletini yıkayın. </li><li> DNase (10 ug / ml) ihtiva eden 500 ul FACS tampon maddesi içinde pelletini ve canlılığı boya kontrol olarak bir kenara 50 ul tablet koydu. </li><li> Seçilen boya için belirtilen konsantrasyonda kalan örnek seçim canlılığı boya ekleyin. </li><li> FACS analizi 31 gerçekleştirin ve Canlılık Dye- / CD31 / CD45- / Tie2- / Ter-119- / EpCAM- hücreleri (Şekil 2A) için sıralama. Sıralama doğrudan FACS tampon içine. </li></ol>

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The authors have nothing to disclose.

Bu yazıda tarif edilen protokol, bir alt seçmek ya da daha sonraki analizler yapılmadan önce, hücre kültüründe bir zaman gerektiren ya mevcut yöntemlere kıyasla, FACS-bazlı sıralama ile fibroblastlar izole etmek için bir araç sunmaktadır. Fibroblast sıralama için cildin hasat gerekli süre yaklaşık 6 saattir; Bununla birlikte, hasat kullanılan farelerin sayısı, bu tahmin etkileyecektir. Protokolde birkaç nokta özellikle dikkat gerektirir. İlk izolasyon prosesi esnas?...

Fibroblastlar sık ​​plastik yüzeylerde uygun iğ biçimli hücreler olarak morfolojik tanımlanır. Fibroblastlar sentezlenmesi ve embriyonik ve yetişkin organlarının 1 hücre dışı matriks remodeling sorumlu ilke hücre tipi vardır. Fibroblastlar memeli gelişme dolayısıyla kritik ve her doku ve organda mevcut hücre tipleri komşu davranışını etkileyen hücre dışı ortamın önemli ölçüde katkıda bulunur. Fibroblastlar de muazzam bir klinik yükünü neden tıbbi koşullar çeşitli bir kümesi arkasında asıl hücre tipi vardır. Patolojik fibroblast aktivitesinin normal doku işlevini bozar ve deri yara iyileşmesi, ateroskleroz, sistemik skleroz, ve damar yaralanması 2-5 sonra aterom plaklarının oluşumuna aşağıdaki doku ve (örneğin akciğer ve karaciğer gibi), organ fibrozis, skar içerir. Özellikle yara iyileşmesi, hem akut ve kronik, d içerirnormal doku gibi ne benzeyen, ne de işlevleri çevreleyen ve çeşitli patolojik devletler arasında önemli morbidite neden skar dokusunun eposition. Travmalarından sonra, daha sonra, yapısal ECM bileşenleri salgılar hücre tiplerini komşu parakrin etkilere neden ve skar dokusu 6 yatırma mekanik istikrarı sağlamak miyofibroblastlar fibroblastların bir geçiş vardır. Deri dokularda gelişimsel süre boyunca yara onarım kalitesi ve anatomik siteler arasında önemli varyasyon mevcuttur. Yaşamın ilk iki trimesterde fetüs iz bırakmadan iyileşir; Ancak, üzerinde ve yetişkinlik boyunca üçüncü trimesterde, bir yara ile iyileşir insanlarda. Mevkiye-özgü yaşa özgü ek olarak, yara iyileşmesi farklılıklar vardır. Deri yaraların içinde skar dokusu biriktirme 9 önemli ise ağız boşluğunda yaralar, minimal skar oluşumu 7,8 ile pişmanlık. Münakaşa c devamyaş ve konum 10,11 hem açısından yara iyileşmesi sonucu yerel fibroblast içsel özelliklerinin karşı çevrenin göreceli etkisini oncerning. Deri dermis ve önceki embriyonik (E15) karşı sözlü fare iyileşmesinde önemli farklılıklar göz önüne alındığında vs sonra embriyonik (E18) dermiş, bazı gelişimsel yaşta ve çeşitli anatomik siteler arasında fibroblastların popülasyonlarında içsel farklılıklar var olasıdır . 1986 yılında, Harold F. Dvorak tümörler 12 iyileşmeyen yaralar vardır oturtulması. Dvorak tümörler vücutta yaralar gibi davranırlar ve konağın yanıtını yaranın iyileşme aktive ederek stroma neden olduğu sonucuna varıldı. Çeşitli çalışmalar beri karsinom 13-15 ilerlemesine fibroblastların katkısını araştırdık, ama yara iyileşmesi, kimlik ve deri ca stromal bölmesine katkıda fibroblast embriyonik kökenli olduğu gibircinomas yeterince tanımlanmamıştır. Bu sorunun cevabı, anti-kanser terapisi 16 için potansiyel olarak etkin hedef olarak tümörle ilişkili fibroblast açığa son çalışmalar verilen tıbbi alaka taşımaktadır. Belirlenmesi ve ileriye dönük in vivo fibrojenik potansiyeli sahip fibroblast soy izole etkin bir akut ve kronik hastalık durumları geniş bir yelpazesinde yaralanma kendi tepkisini manipüle yönelik önemli bir adımdır. 1987 yılında, Cormack papiller ve retiküler dermis 17,18 içinde biri içinde ikamet eden fibroblast iki alt popülasyonlarını bir gösterdi. Üçüncü ICSI&#39;nin follikül 19,20 Dermal papilla bölgedeki saç köklerinin ile ilişkili bulunmuştur. Ne zaman kültür, büyüme potansiyeli, morfoloji ve büyüme faktörü / sitokin bu fibroblast alt tiplerinin sergi farkları 21-24 profilleri. Bugüne kadar, fibroblast inceleyen çalışmaların oterogeneity ölçüde yeterli in vivo fibroblastlar arasında gelişimsel ve fonksiyonel çeşitlilik karakterize etmek başarısız oldu. Bu, kısmen, kültürlenmiş fibroblast popülasyonları bir güven ve tüm fibroblastlar 25 ile ifade edilen bir kendi kendine yüzey reseptörüne göre hücre kültüründen ya da pozitif bir seçim homojenize etkisinin bir sonucu olduğu. Laboratuarımızda yapılan yeni bir çalışma, derin bir yüzey işaretleyici ve bu yazıda 26 sunulan FACS-bazlı izolasyon metodolojisi ile izole vs kültürlü kültürsüz fibroblast transkripsiyonel vardiya gösterdi. Daha sonra, kemirgen dorsal dermiş içinde belirli bir fibroblast soy tanımlanmış ve Engrailed-1 embriyonik ekspresyonu ile tanımlanan, bu soy, dorsal, derideki bağ dokusu birikimi için temel olarak sorumlu olduğu tespit edilmiştir. Yara iyileşmesi, kanser stroma oluşumu da dahil olmak üzere fibrozis, akut ve kronik biçimleri boyunca da soy fonksiyonlarıRadyasyon fibrozis 27 uyarılan. Farklı fibroblast soy karakterizasyonu fibrojenik davranışlarını modüle amaçlayan tedaviler için kritik etkileri vardır. Aksine, hücre izolasyonu 28,29 ulaşmak için in vitro manipülasyon güvenmek mevcut protokolleri kullanarak daha burada ayrıntılı hasat protokolü (Şekil 1) daha doğru in vivo fenotip ve davranışlarını yakalamak fibroblast verimi bilgilendirici analizlerini yardımcı olacaktır.

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			<td height="21" style="height:21px;">Ammonium-Chloride-Potassium (ACK) lysing buffer</td>
			<td>Gibco</td>
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			<td>Fisher Scientific</td>
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			<td>BioLegend</td>
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			<td height="17" style="height:17px;">Ter-119</td>
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			<td height="21" style="height:21px;">propidium iodide (PI viability stain)</td>
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murine dermal fibroblast isolation by facs

Fibroblastlar hücre dışı matriks salgılayan sorumlu ilke hücre tipi ve birçok organ ve dokuların kritik bir bileşenidir. Fibroblast fizyolojisi ve patolojisi birden organlarda fibrozların, hipertrofik skar aşağıdaki yanıklar, iskemi sonrası kardiyak fonksiyon kaybı ve kanser stroma oluşumu da dahil olmak üzere klinik kuruluşların, bir spektrum altında yatan. Ancak, fibroblastlar büyük ölçüde kendi içsel heterojenite, hücrenin kötü karakterize tip kalır. Fibroblastlar izolasyonu için mevcut yöntemler derinden hücre fenotipi ve davranışlarını etkileyen hücre kültüründe zaman gerektirir. Sonuç olarak, fibroblast biyoloji araştıran birçok çalışma, in vitro manipülasyon güvenmek ve doğru bir in vivo fibroblast davranışı yakalamak değil. Bu problemin üstesinden gelmek için, preservi böylece, hücre kültürü gerektirmez, yetişkin farelerin dorsal deri fibroblast izolasyonu için bir FACS tabanlı bir protokol geliştirilmiştirfizyolojik transkripsiyonel ve her hücrenin proteomik profili ng. Bizim stratejimiz soy negatif kapısı yoluyla olmayan mezenkimal soy dışlama sağlar (Lin -) oldukça spesifik yüzey belirteçleri ifade fibroblastlar bir alt ön seçim veya zenginleştirme önlemek ve bu heterojen genelinde mümkün olduğunca kapsayıcı olması için pozitif bir seçim stratejisi daha hücre tipi.

Bu yaklaşım (Şekil 1) geçerlilik son yayın 27 ayrıntılı incelenebilir şekilde, bir dizi doğrulanmıştır. Bunlar sıralanmış hücreler ve kitle hücre ve taze sıralanmış hücrelerin tek bir hücre transkripsiyonel analizi immünsitokimya içerir. Fibroblastlar Sıralama doğrudan ziyade daha doğru kültürüne dayanarak in vivo fenotip yakalar. Yerine olumlu bir seçim yaklaşımı daha soy negatif tüketme yaklaşımı (Şekil 2A) kullanılarak belirli alt popülasyonla...

Watch this Scientific Journal Video about Murine Dermal Fibroblast Isolation by FACS at JoVE.com

Murine Dermal Fibroblast Isolation by FACS

FACS ile Murin Dermal fibroblast İzolasyonu

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

murine-dermal-fibroblast-isolation-by-facs

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

20.9K Görüntüleme.  Stanford University School of Medicine. Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Video: Murine Dermal Fibroblast Isolation by FACS

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Nöral Analizi Yetişkin Fare V-SVZ&#39;una Hücre istikrarlı ve verimli Genetik Modifikasyon Kök Hücre Özerk ve Non-özerk Etkileri

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Gelişim Biyolojisi

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Fare Kulak ve Kuyruk Dokular Yaratma İlköğretim Fibroblast Kültürleri

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

İnsan Frozen Gastrointestinal örneklerden CD 90+ fibroblast / miyofibroblastlar İzolasyonu


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Methods to Study Mrp4-containing Macromolecular Complexes in the Regulation of Fibroblast Migration

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of cyclic nucleotides. By modulating intracellular cyclic nucleotide concentration, MRP4 can regulate multiple cyclic nucleotide-dependent cellular events including cell migration. Previously, we demonstrated that in the absence of MRP4, fibroblast cells contain higher levels of intracellular cyclic nucleotides and can migrate faster. To understand the underlying mechanisms of this finding, we adopted a direct yet multifaceted approach. First, we isolated potential interacting protein complexes of MRP4 from a MRP4 over-expression cell system using immunoprecipitation followed by mass-spectrometry. After identifying unique proteins in the MRP4 interactome, we utilized Ingenuity Pathway Analysis (IPA) to explore the role of these protein-protein interactions in the context of signal transduction. We elucidated the potential role of the MRP4 protein complex in cell migration and identified F-actin as a major mediator of the effect of MRP4 on cell migration. This study also emphasized the role of cAMP and cGMP as key players in the migratory phenomena. Using high-content microscopy, we performed cell-migration assays and observed that the effect of MRP4 on fibroblast migration is completely abolished by disruption of the actin cytoskeleton or inhibition of cAMP-dependent kinase A (PKA). To visualize signaling modulations in a migrating cell in real time, we utilized a FRET-based sensor for measuring PKA activity and found, the presence of more polarized PKA activity near the leading edge of migrating Mrp4-/- fibroblast, compared to Mrp4+/+fibroblasts. This in turn increased cortical actin formation and augmented the process of migration. Our approach enables identification of the proteins acting downstream to MRP4 and provides us with an overview of the mechanism involved in MRP4-dependent regulation of fibroblast migration.

MRP4 regulates various cyclic nucleotide-dependent signaling events including a recently elucidated role in cell migration. We describe a direct, but multifaceted approach to unravel the downstream molecular targets of MRP4 resulting in identification of a unique MRP4 interactome that plays key roles in the fine-tuned regulation of fibroblast migration.

Yöntemler Fibroblast Göç Yönetmeliği Mrp4 içeren makromoleküler kompleksler Eğitim için

Multidrug resistance protein 4 (MRP4) is a member of the ATP-binding cassette family of membrane transporters and is an endogenous efflux transporter of ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

Romatoid Artrit Hastalarının Mafsallar İzole fibroblast benzeri sinoviyosit kullanma Kaynaklı-pluripotent Kök Hücre Üretimi

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

Fare Embriyonik Hemogenic endotel hücrelerinin izolasyonu

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

Isolation and Enrichment of Liver Progenitor Subsets Identified by a Novel Surface Marker Combination

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular infiltrate and epithelial cell activation. The progenitor cell population during such inflammatory reactions has mostly been investigated using single surface markers, either by histological analysis or by flow cytometry-based techniques. However, novel surface markers identified various functionally distinct subsets within the liver progenitor/stem cell compartment. The method presented here describes the isolation and detailed flow cytometry analysis of progenitor subsets using novel surface marker combinations. Moreover, it demonstrates how the various progenitor cell subsets can be isolated with high purity using automated magnetic and FACS sorting-based methods. Importantly, novel and simplified enzymatic dissociation of the liver allows for the isolation of these rare cell populations with a high viability that is superior in comparison to other existing methods. This is especially relevant for further studying progenitor cells in vitro or for isolating high-quality RNA to analyze the gene expression profile.

Liver injuries are accompanied by progenitor cell expansion that represents a heterogeneous cell population. Novel classification of this cellular compartment allows for the distinguishing of multiple subsets. The method described here illustrates the flow cytometry analysis and high purity isolation of various subsets that can be used for further assays.

İzolasyon ve Roman Yüzey Etiket Kombinasyon ile tanımlanır Karaciğer progenitör Alt Grupları zenginleştirilmesi

During chronic liver injuries, progenitor cells expand in a process called ductular reaction, which also entails the appearance of inflammatory cellular ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Lineage yetişkin fare Fibroblast Erythroid ataları için yeniden programlama doğrudan

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Isolation of Murine Spermatogenic Cells using a Violet-Excited Cell-Permeable DNA Binding Dye

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established cell isolation protocols using Hoechst 33342 staining in combination with fluorescence-activated cell sorting, it requires cell sorters equipped with an ultraviolet laser. Here we describe a cell isolation protocol using the DyeCycle Violet (DCV) stain, a low cytotoxicity DNA binding dye structurally similar to Hoechst 33342. DCV can be excited by both ultraviolet and violet lasers, which improves the flexibility of equipment choice, including a cell sorter not equipped with an ultraviolet laser. Using this protocol, one can isolate three live-cell subpopulations in meiotic prophase I, including leptotene/zygotene, pachytene, and diplotene spermatocytes, as well as post-meiotic round spermatids. We also describe a protocol to prepare single-cell suspension from mouse testes. Overall, the procedure requires a short time to complete (4-5 hours depending on the number of needed cells), which facilitates many downstream applications.

Here we present a simple and efficient method to isolate live meiotic and post-meiotic germ cells from adult mouse testes. Using a low-cytotoxicity, violet-excited DNA binding dye and fluorescence-activated cell sorting, one can isolate highly enriched spermatogenic cell populations for many downstream applications.

Mor-Heyecanlı Hücre-Geçirgen DNA Bağlayıcı Boya Kullanılarak Murine Spermatojenik Hücrelerin İzolasyon

Isolation of meiotic spermatocytes is essential to investigate molecular mechanisms underlying meiosis and spermatogenesis. Although there are established ...

Isolation of Murine Retinal Endothelial Cells for Next-Generation Sequencing

Recent improvements in next-generation sequencing have advanced researchers&#39; knowledge of molecular and cellular biology, with several studies revealing novel paradigms in vascular biology. Applying these methods to models of vascular development requires the optimization of cell isolation techniques from embryonic and postnatal tissues. Cell yield, viability, and purity all need to be maximal to obtain accurate and reproducible results from next-generation sequencing approaches. The neonatal mouse retinal vascularization model is used by researchers to study mechanisms of vascular development. Researchers have used this model to investigate mechanisms of angiogenesis and arterial-venous fate specification during blood vessel formation and maturation. Applying next-generation sequencing techniques to study the retinal vascular development model requires optimization of a method for the isolation of retinal endothelial cells that maximizes cell yield, viability, and purity. This protocol describes a method for murine retinal tissue isolation, digestion, and purification using fluorescence-activated cell sorting (FACS). The results indicate that the FACS-purified CD31+/CD45- endothelial cell population is highly enriched for endothelial cell gene expression and exhibits no change in viability for 60 min post-FACS. Included are representative results of next-generation sequencing approaches on endothelial cells isolated using this method, including bulk RNA sequencing and single-cell RNA sequencing, demonstrating that this method for retinal endothelial cell isolation is compatible with next-generation sequencing applications. This method of retinal endothelial cell isolation will allow for advanced sequencing techniques to reveal novel mechanisms of vascular development.

This protocol describes a method for the isolation of murine postnatal retinal endothelial cells optimized for cell yield, purity, and viability. These cells are suitable for next-generation sequencing approaches.

FACS ile Murin Dermal fibroblast İzolasyonu

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Nöral Analizi Yetişkin Fare V-SVZ'una Hücre istikrarlı ve verimli Genetik Modifikasyon Kök Hücre Özerk ve Non-özerk Etkileri