Yazarlar Janet Rossant orijinal protokol için teşekkür ederiz. Bu çalışma Ulusal Sağlık hibe CA106308 WH Enstitüsü tarafından desteklenmiştir.

Bu protokol, fare blastosist hazırlanması ve TS hücre hatlarının kurulması açıklanmaktadır. Doğal çiftleşme için set ve süper ovulasyon indüksiyon dahil olmak üzere blastosist koleksiyon, önce Genel fare manipülasyonlar temelde Nagy ve ark tarafından gösterilen standart bir protokol izleyin. 2003 (pp146 150). TS hücrelerinin türetilmesi ve bakım <ol><li> Kur, faiz fareler arasında üreme çapraz. </li><li> Besleyici hücreler olarak fare embriyonik fibroblastlar (MEFS) hazırlayın. Blastosist koleksiyon İki gün önce, MEFS (2 x 10 6 hücre) 100 mm yemekler kaplanmıştır. Ertesi gün, bu hücrelerin, 37 ° C,% 5 CO 2 saat inkübasyon için 2 mitomisin C (20 mg / ml) içeren 10 ml TS orta ile tedavi edilir . Bu hücreler PBS ile iki kez yıkanarak takip eder. Mitomisin C muamele edilen hücreler, daha sonra 12 kuyucuğu (1 x 10 5 hücre / iyi) numaralı seribaşı. </li><li> Blastosist toplama (3,5 DPC, bir gün olarak anılacaktır) günü, MEF kültür ortamı TS orta artı 1x FGF4/Heparin (0.5 ml / kuyu) tarafından değiştirilir. </li><li> Rahim toplanması için hazırlık: fare euthanize ve rahim (Şekil 1A) bulun. </li><li> Çıkarın, serviks (mesane arkasında bulunan) forseps ile tutarak rahim ve rahim ve serviks (Şekil 1B) birleşme kesen. Rahim çekin uzak membran ve yağ dokuları Döşeme ve yumurtalık (Şekil 1C) ile birleşme altında rahim kesmek. TS orta küçük bir hacmi (0,2 ml) 60 mm plaka uteri (Şekil 1D) yerleştirin. </li><li> 1 ml 26 gauge iğne ile 1 ml şırınga kullanarak TS orta ile her uterus boynuzu yıkayın. Iğne rahim tabanı içine yerleştirin ve yavaş yavaş yeni bir 60 mm plakası içine 1 ml TS orta ile yıkayın. Rahim şişirilmiş ve kızarma sırasında tamamen kapsayacak şekilde genişletilmesi gerekmektedir. </li><li> Toplayın ve bir ağız kontrollü pipet kullanarak TS orta içeren temiz bir 12 plaka dikkatlice blastosist transferi. 12 kuyucuğu TS aracılığıyla seri transfer blastosist üç kez yıkayın. Bir de ortalama 37 mitomisin C-MEF besleyiciler tedavi ve kültürü içeren bir 12-plaka blastosist (Şekil 2A) ° C,% 5 CO 2. </li><li> Blastosist zona pellusida yumurtadan çıkar ve 24 ila 36 saat içinde kuyulara eklenecektir. Küçük bir akıbet 3. gününde kolaylıkla görülecektir. 500 ul taze TS orta artı 1x FGF4/Heparin kültürler besleyin. </li><li> 4 veya 5 günde, blastokist akıbet boyutuna bağlı olarak ayrıştırılmasıyla için hazır olmalıdır. TH hücre ayrıştırılmasıyla için ideal boyut Şekil 3B gösterilmiştir. Kültürlerin 0.5 ml PBS ile bir kez yıkayın. 5 dakika 37 ° ° C,% 5 CO 2 için 100 ml% 0.25 tripsin / EDTA ve inkübe ekleyin. P100 pipetteman ile kuvvetlice yukarı ve aşağı pipetleme küçük öbekler halinde Break sonucudur. TS-70cm-1.5F4H (% 30 TS orta,% 70 MEF-CM artı 1.5x FGF4/Heparin) kuyunun içine ekleyerek trypsinization durdurun. Ayrıştırılmasıyla 8 saat sonra orta değiştirin ve her 2 günde hücreleri beslemeye devam. </li><li> 6 ve 10 gün (son derece değişken) arasında TS hücre kolonileri görülebilir. Onlar açık bir koloni sınır (Şekil 2C) morfoloji gibi yassı epitel levha var. 2 günde kültürlerin beslemeye devam </li><li> TS hücreleri% 50 confluency (genellikle 15 ve 20 gün arasında) ulaştığınızda, PBS ile bir kez yıkayın ve 100 ml% 0.25 tripsin / EDTA ekleyin. Trypsinization sırasında yakın bir tek hücre süspansiyonu sağlamak için yukarı ve aşağı Pipet. Mitomisin C-MEF besleyiciler tedavi (2.5 x 10 5 cells/6-well veya 5 x10 5 cells/60mm plaka) içeren 6-iyi ya da 60mm plakalar yeni TS-70cm-1.5F4H ve transfer ekleyerek trypsinization durdurun. </li><li> Değişim ve orta 8 saat geçtikten sonra 2 gün her kültürleri beslemeye devam edin. </li><li> MEF besleyiciler bir veya iki pasajlar daha sonra (3-5 gün pasajlar arasında), TS hücreleri onlar olmadan kültür olabilir. MEFS trypsinization sonra TS hücrelerden daha çok daha hızlı kültür plaka takmak Çünkü, yapışık oranı böyle bir fark MEF besleyiciler TS hücreleri ayırmak için kullanılabilir. Kültür hücreleri geçtikten sonra 37 dakika süreyle 30 ° C,% 5 CO 2 kuluçkaya yatmaktadır. En MEFS plaka takmak ve TS nüfus süspansiyon kalır. Yeni bir plaka süpernatantı aktarın. Saf bir TS nüfus elde etmek için çeşitli pasajlar bu işlemi tekrarlayın. </li><li> TS-70cm-1.5F4H TS hücreleri korumak ve her 4 gün geçmesine (1:10 - 1:20 bölünmüş) ya da kültürün% 70 confluency ulaşır. Yüksek yoğunluklu (01:05 bölünme daha yüksek) aşırı konfluent farklılaşma neden olabilir ya da kültür hücreleri geliyorsunuz. </li><li> TS hücrelerinin kimliğini bir hücre belirteci, Cdx2 (Şekil 3) immün teyit edilebilir. </li></ol> Donma TS hücreleri <ol><li> Dondurma orta olarak (% 50 FBS,% 30 TS orta ve% 20 DMSO) 2x hazırlayın. </li><li> Cotrypsinization tarafından llect TS hücreleri, 6 dakika süreyle santrifüj 450g. 2x dondurma orta eşit hacmi ile TS orta onları tekrar </li><li> , Yavaş yavaş -70, izopropil alkol odası kullanarak hücrelerin Freeze ° C ve 24-48 saat sonra sıvı azot transfer. </li><li> (Confluent% 70) 60 mm plaka genellikle 2 şişeleri tutulur. </li></ol> TS hücreleri Çözülme <ol><li> Flakon çözünme hızla 37 ° C, (450g, 6 dakika) santrifüj tarafından 10 ml TS medya onları toplamak ve% 30 TS medya, 70cm 1.5F4H tekrar süspansiyon haline getirin. </li><li> Hücrelerin Bir flakon, 60 mm plaka numaralı seribaşı. </li></ol> TGC içine TS hücre farklılaşması Trofoblast dev hücreler (TGCs) içine TS hücrelerin farklılaşması, MEF-CM, FGF4 ve heparin çekilmesi ile indüklenen olabilir. TGC fenotip (büyük çekirdekleri), dört gün indüksiyon sonra tespit edilebilir (Tanaka ve ark, 1998 ve Chiu ve ark 2008) . TGC farklılaştırılmış kültürü bu işaretleyici (Şekil 4A) p450scc, immün ifade algılar. PI Flow sitometrik analizi (propidium iyodür) boyanan hücreler daha yüksek DNA içeriği (Şekil 4B) içeren farklılaşmış hücrelerin varlığı gösterir. Bu sonuçlar, TS hücreleri polyploid TGCs haline endoreduplication geçmelerini öneriyoruz. Temsilcisi Sonuçlar <img src="/files/ftp_upload/1964/1964fig1.jpg" alt="Şekil 1" /> Şekil 1 fare uteri Diseksiyon. (A) uteri yeri oklarla gösterilir. (B) gösterildiği gibi, rahim ve serviks kavşak boyunca kesin. (C), over (OV) altında yumurtalık (OD) altında rahim kesin. Uteri (D) Görüntü. <img src="/files/ftp_upload/1964/1964fig2.jpg" alt="Şekil 2" /> Şekil 2 Temsilcisi resimleri fare blastosist, blastosist akıbet ve trofoblast koloni göstermektedir. (A) E3.5 toplanan bir fare blastosist gösterilmiştir. ICM, iç hücre kütlesi MT, duvar trophectoderm; PT, kutup trophectoderm. (B) MEF besleyiciler trofoblast akıbet gösterilmiştir. TS, trofoblast kök hücre. (C) trofoblast kök hücre kolonisi MEF besleyiciler açık bir kenar gösterir. Oklar hücre sınırı göstermektedir. <img src="/files/ftp_upload/1964/1964fig3.jpg" alt="Şekil 3" /> Şekil 3 analiz immün TS hücrelerinin belirlenmesi. TS (AD) ve ES (kontrol, EH) hücreleri işaretleyici kimlik tabi. Hücreler Oct4 (A, E) ya da Cdx2 (B, F) tanımak için antikorlar ile boyanmış ve birleştirilmiş görüntüler sunulan DAPI (C, G) (D, H) tarafından zıt. Ölçeği bar, 50 mm. <img src="/files/ftp_upload/1964/1964fig4.jpg" alt="Şekil 4" /> Şekil 4 TS hücrelerinin in vitro Farklılaşma. (A) kültür ayrım yapılmamış (Undiff) veya farklılaştırılmış (Diff) hücreleri, TGC bir marker ifade için incelenmiştir p450scc (yeşil), DAPI (kırmızı) ile zıt. (B) PI ile boyandı farklılaşmış hücrelerin, flow sitometrik analizi, DNA içeriği (M2, dörtten fazla kopya M1, 03:58 kopya) ölçer. M2 nüfusu polyploid trofoblast hücreleri temsil eder.

<ol>
	<li>Chiu, S. Y., Asai, N., Costantini, F. &. a. m. p. ;. a. m. p., Hsu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SUMO-specific+protease+2+is+essential+for+modulating+p53-Mdm2+in+development+of+trophoblast+stem+cell+niches+and+lineages.">SUMO-specific protease 2 is essential for modulating p53-Mdm2 in development of trophoblast stem cell niches and lineages.</a> PLoS Biol. 6, E310-E310 (2008).</li><li>Kunath, T., Arnaud, D., Uy, D. G., Okamoto, I., Chureau, C., Yamanaka, Y., Heard, E., Gardner, R. L., Avner, P., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imprinted+X-inactivation+in+extra-embryonic+endoderm+cell+lines+from+mouse+blastocysts.">Imprinted X-inactivation in extra-embryonic endoderm cell lines from mouse blastocysts.</a> Development. 132, 1649-1661 (2005).</li><li>Nagy, A., Gertsenstein, M., Vintersten, K., Behringer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manipulating the Mouse Embryo: The Laboratory Manual. , (2003).</li><li>Tanaka, S., Kunath, T., Hadjantonakis, A. K., Nagy, A., Rossant, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Promotion+of+trophoblast+stem+cell+proliferation+by+FGF4.">Promotion of trophoblast stem cell proliferation by FGF4.</a> Science. 282, 2072-2075 (1998).</li></ol>

Bu videoda, biz süreci TS hücre hatları kurmak için uteri ve deneysel prosedürleri E3.5 blastosist toplamak göstermektedir. Ayrıca stemness TS hücreleri korumak ve farklılaşmış hücrelerine farklılaşma ikna etmek için durumu açıklar. Saf TS hücre hatları elde etmek için iki kritik adımlar akıbet ayrıştırılmasıyla için zamanlama (adım 9) ve ilk geçiş işleme (adım 11). Bu geniş blastosist adım 11 (Kunath ve ark, 2005) TS kolonilerin adım 9 ya da aşırı çoğalma (&gt;% 50 confluency) ...

Reaktifler TS orta: RPMI-1640,% 20 FBS, 1 mM sodyum piruvat, 100 mM beta-merkaptoetanol ve 100 mg / ml penisilin-streptomisin. Mitomisin C: 2 mg mitomisin C (Sigma M-0503) 2 ml PBS içinde çözülür ve 200 ml TS orta (10 ng / ml) 2 ml içine bu karışımı ekleyin. Kullanana kadar -20 ° C&#39;de 20 alikotları (10 ml) ve mağaza olun. 100 mm plaka başına bir kısım ekleyin. Fare embriyonik fibroblast koşul orta (MEF-CM): 100 mm plakalar Levha MEFS (1 x 10 6 / plaka). Ertesi gün başına 10 ml TS çanak orta ekleyin ve 48 saat boyunca kültür devam ediyor. Bunları filtre (0.45 mm) ve 35 ml alikotları -20 ° C&#39;de mağaza, kültür ortamı toplayın. 4 saklanabilir gerekli her kısım ° C çözülme MEFS konfluent haline gelmeden CM iki veya daha fazla partiler için hazırlamak için kullanılabilir. PBS / BSA: 0.45 mm şırınga filtresi ile filtre PBS (10 ml), BSA, fraksiyon V (Sigma A3311,% 0,1 (w / v)) çözülür ve 1 ml 1,5 ml tüpler alikotları yapmak. Mağaza -70 ° C ve FGF4 hazırlamak için gerekli zaman bir tüp Çözülme. FGF4 (1000x): 1 ml PBS / BSA çözüm ile yeniden süspanse liyofilize FGF4 (Sigma F8424, 25 mg). -70 Iyice karıştırınız ve 1.5 ml tüpler ve depoya 10 alikotları (100 ml) ° C Gerektiğinde her tüp çözülme ve saklamak kalan 4 ° C, tekrar dondurmak yok. 1x FGF4 (25 ng / ml) elde etmek için, TS orta 1000 kat sulandırınız. Heparin (1000x): 1 nihai konsantrasyonu PBS içinde süspanse edin heparin (Sigma H3149, 10.000 adet) mg / ml (1000x). 1.5 ml tüpler ve mağaza içine 100 ml alikotları olun -70 ° C Çözülme alikotları ve kalan 4 saklamak ° C, yeniden dondurucu yoktur. 1x heparin (1 ng / ml) elde etmek için, TS orta 1000 kez sulandırınız.

derivation of mouse trophoblast stem cells from blastocysts

Trophectoderm belirlenmesi memeli gelişiminin erken farklılaşma olayları biridir. Trophectoderm aracılık implantasyon türetilmiştir ve plasentanın fetal trofoblast soy üretir. Sonuç olarak, bu soy gelişimi embriyonun hayatta kalmak için çok önemlidir. Fare blastosist trofoblast kök (TS) hücreleri türetilmesi ilk Tanaka tarafından tarif edilmiştir<em&gt; Ve ark.</em&gt; 1998. Uygun stimülasyon sonra sahne ve hücre tipi özel işaretleri trofoblast özel mülkiyet ve anlatım TS hücreleri korumak için yeteneği erken plasenta olayları yansıtan sayede trofoblast soy geliştirme araştırmak için değerli bir model sistem sunmaktadır. Ayrıca, trofoblast hücrelerinin doğal genom amplifikasyon geçiren birkaç somatik hücre tipleri biri. Trofoblast polyploidization altında yatan moleküler yollar çözülmeye başlamış olmasına rağmen, trofoblast genom amplifikasyon fizyolojik rolü ve avantajı büyük ölçüde zor kalır. Kültür polyploid trofoblast hücreleri diploid kök hücre gelişimi bu<em&gt; Ex vivo</em&gt; Sistem, sağlık ve hastalık, genom replikasyonu ve istikrarsızlık düzenleyici bir mekanizma nedenlerine açıklık için mükemmel bir araç. Burada Chiu yılında yayımlanan değişiklik ile önceki raporlara göre bir protokol<em&gt; Ve ark.</em&gt; 2008.

Watch this Scientific Journal Video about Derivation of Mouse Trophoblast Stem Cells from Blastocysts at JoVE.com

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Bu video, fare blastosist blastosist trofoblast kök hücrelerinin izolasyonu ve derivasyon göstermektedir. Ayrıca kök hücre özelliği yanı sıra kültür farklılaşma indüksiyonu için bakım koşulları açıklar.

Blastosist Fare trofoblast Kök Hücre türetilmesi

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the ...

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18.1K Görüntüleme.  University of Rochester. Bu video, fare blastosist blastosist trofoblast kök hücrelerinin izolasyonu ve derivasyon göstermektedir. Ayrıca kök hücre özelliği yanı sıra kültür farklılaşma indüksiyonu için bakım koşulları açıklar.

Video: Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

Murin Embriyonik Kök Hücre Hematopoetik Kök Hücre türetilmesi

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

Biyoloji

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

Immunosurgery İnsan Embriyonik Kök Hücreleri türetilmesi

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Fare Tükürük Bezi Kök Hücre İzolasyonu

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

Birincil Fare trofoblast hücreleri ve trofoblast invazyonu Assay İzolasyonu

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Küçük Molekül İndüksiyon Pluripotent İnsan Embriyonik Kök Hücreleri İnsan Kardiyak Öncüleri ve kardiyomiyositlerde Verimli türetilmesi

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

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 ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Uzun vadeli Yetişkin Fare SSC Hatları türetilmesi için Kültür Spermatogonial Kök ve progenitör hücrelerin Seri Zenginleştirme (DGM)

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

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 ...