1. Embriyo kaldırma ve plasental diseksiyonu <ol><li> Başlamak için, embriyolarının ilk hamile barajlardan izole edilmelidir. </li><li> Hayvanlar onaylanmış prosedürlere göre ötenazi bizim durumumuzda ölümcül dozda anestezi izofluran kullanmak için gidiyoruz </li><li> İlk olarak, etanol ile karın sprey ve bir makas ile küçük bir kesi yapmak. </li><li> Karın ortaya çıkarmak ve periton açık kesmek için iki elinizle deri koparmak. </li><li> Forseps ile uterin boynuz pick up ve onları her distal sonunda makas kullanarak toplamak. PBS ve yer buz dolu bir petri rahim boynuzları yerleştirin. Ayrıca, plasenta izole önce PBS ile birkaç kez yıkamak için hatırlıyorum. Biz şimdi plasenta ayırabilirsiniz. </li><li> Iki forseps ile embriyo conceptuses çevreleyen endometrial doku dikkatlice uzakta soyma başlar. Bir delinme veya embriyoların yapısal hasar, aksi takdirde sonraki diseksiyonu adımları zorlaştıracaktır dikkatli olmalıdır. </li><li> PBS ile buz üzerinde yeni bir petri izole conceptuses yerleştirin ve tüm conceptuses endometrium kurtulmuş kadar devam ediyor. </li><li> PBS ile mikroskop altında ve iki forseps plasenta desidua kabuğu ile yerleştirilen yeni bir petri bir konseptus aktarın. Desidua hücreleri tek hücre süspansiyonu ve flow sitometri ile müdahale mümkün olduğunca desidua kadar çıkarın. Pürüzsüz sürekli soyma hareketi en iyi sonuçları elde etmek için tavsiye edilir. </li><li> Iki organ arasında kavşakta plasenta yolk kesesi uzak kesin ve nazikçe plasenta uzak yolk kesesi ve vitellin damarları çekin. Bakım, özellikle erken embriyolar, plasentanın koryonik plaka bozmak için değil alınmalıdır. </li><li> Forseps çifti ve başka bir ile göbek kordonu ile plasentanın koryonik plaka dikkatlice tutarak, göbek kordonunun plasenta uzak ve bağlı embriyo çekin. </li><li> Tek hücre süspansiyonu hazırlanması sırasında topaklanma hücre en aza indirmek için plasentanın kenarlarında aşırı dev hücre dokusu çıkarın. </li><li> PBS +5% FCS ve buz üzerinde, uygun bir kaba 15 ml Falcon tüp gibi, plasenta toplayın. Disseke tüm plasenta için bunu yapın. </li><li> Bu noktada, ya da in vitro kültür veya transplantasyon gibi flow sitometri, fonksiyonel testleri, tek bir hücre süspansiyonu hazırlanması ile devam edebilirsiniz ya da doku fiksasyon ve nihai immünohistokimya için gömme sabit dondurulmuş blok için bütün plasenta hazırlamak. </li></ol> 2. Plasenta dokusunun tek hücre süspansiyonu hazırlanması <ol><li> Şimdi FACS analizi için plasentanın tek bir hücre süspansiyonu nasıl oluşturmak için size gösterecektir. </li><li> Plasenta tek bir hücre süspansiyonu, mekanik ve enzimatik ayrılma hazırlamak için gereklidir. İlk% 10 FCS ve% 1 penisilin / Streptomisin çözüm PBS içinde% 0,1 kollajenaz solüsyon hazırlanır. </li><li> Kaç bağlı olarak kollajenaz çözüm uygun bir hacmi, plasenta ekleyin. Plasenta hacmi ve 2-5ml arasında iki kez hacmi önerilir. </li><li> 5 ml şırınga, iğne 3 kez kollajenaz çözüm ve plasenta yoluyla Pasajlanması doku mekanik bozmaya donatılmış bir 16-G iğne kullanın. 18-G bir iğne ile tekrarlayın. </li><li> 45 dakika boyunca 37 ° C,% 5 CO2 inkübatör örnek yerleştirin. </li><li> 20 G iğne ile kollajenaz, pasajı hücre çözümü, 37, ek bir 45 dakika boyunca inkübe ° C de 45 dakika inkübasyondan sonra </li><li> Her bir iğne ile 22-G ve 25 G iğneler aracılığıyla kollajenaz, pasajı hücre çözümü toplam 1.5 saat inkübasyon sonra 3 kez. </li><li> 50 mikron filtre ile yeni bir 15 ml Falcon tüp takılı bir örnek Filtre, 4 PBS +% 5 FCS ve santrifüj ile yıkayın ° C 5 dakika boyunca 300 × g. </li><li> Bu noktada, biz FACS analiz ve fonksiyonel testleri için uygun tek bir hücre süspansiyonu var. </li></ol> 3. Dondurulmuş Bölüm Doku Fixation <ol><li> Hemen her embriyonun plasenta izole sonra, soğuk 1X PBS yerleştirilir. Genellikle 96-plaka genç plasenta (E8.5-11.5) için en iyi çalışır, ancak büyük plasentası ile E12.5 ve yaşlı, 24 plaka daha uygun olabilir. </li><li> Her plasenta taze çözülmüş% 4 paraformaldahyde veya PFA ile de dolu yeni bir aktarılır. E12.5 plasenta, doku yeni bir de elle taşınır, ancak E10.5 bir pipet yardımıyla dikkatlice PBS kaldırmak ve PFA ekleyin. Plasenta doku yaşına ve büyüklüğüne göre tamamen, 4 ° C&#39;de 2-4 saat süreyle dalmış olduğu önemlidir. </li><li> % 30 sukroz doku transferi, ya da dokuların çok küçük ve narin ise, PFA aspirat ve sakaroz doğrudan ekleyin. Bu aşamada, plasenta çözüm yüzen olacaktır. Dokular 4 geceleme ° C</li><li> % 30 sukroz gecede adım sonra, sonuçta dokuları uygun kriyoprezervasyon göstergesidir iyi alt lavabo olmalıdır. Sakaroz çözeltisini yarısını çıkarın ve OCT ile ses değiştirin ve 4 ° C&#39;de 1-2 saat süreyle doku yerleştirmek. Bu Ekim penetrasyonu kolaylaştırır. </li><li> Gömülü olması, bu tarihten sonra 1 saat süreyle% 100 Ekim doku aktarın. </li></ol> 4. Sabit Doku gömülmesi <ol><li> Uygun doku tanımlama ile (show örnek kalıp) Etiket plastik kalıplar. </li><li> OCT çözüm dokusu dışarı doğru çekin ve disk şeklindeki plasenta göbek kordonu yüzü aşağı bakacak şekilde yönlendirilmesi ve dikkatli bir şekilde sağlamak için baskı bırakmadan önce temiz bir jilet bıçağı ileri geri hareket etmek için emin olun dilimleme yarısında plasenta kesmek bile kesti. Plasenta, daha sonra plasental HKH&#39;lerin görselleştirmek için en iyi prientation ulaşmak için yarıya. </li><li> Bir kalıbın alt yüzeyi ile temas halinde kesme kenarı ile kalıp altındaki plasenta yarım. Diğer yarısı için yeni bir kalıp ile tekrarlayın. E10.5 plasenta için, her iki devreyi de aynı tekniği kullanarak aynı kalıp içine konabilir. </li><li> , Kalıp içine OCT dökülerek zaman, doğru yönde plasenta tutmak zor olabilir. Doku forseps ile yarım tutarak yerde plasenta sabitleyin. Ekim çözüm kalıp içine yavaşça dökün ve varsa kabarcıkları kaçının. E10.5 plasenta, forseps bir doku yarısını tutun ve forseps dışında diğer doku karşı yarım bekletin. Ekim çözüm E12.5 plasenta açıklandığı gibi yavaşça dökün. </li><li> Kalıp OCT ile doldurulduktan sonra, hızlı bir şekilde kuru buz üzerine kalıp, OCT doku dondurma beyaz flaş dönecek </li><li> Kalıp Drierite içeren küçük bir plastik torba içine koyun ve hemen -80 ° C derin dondurucuda saklayın. </li></ol>

<ol>
	<li>Gekas, C., Dieterlen-Lievre, F., Orkin, S. H., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+placenta+is+a+niche+for+hematopoietic+stem+cells.">The placenta is a niche for hematopoietic stem cells.</a> Dev Cell. 8, 365-375 (2005).</li><li>Rhodes, K. E., Gekas, C., Wang, Y., Lux, C. T., Francis, C. S., Chan, D. N., Conway, S., Orkin, S. H., Yoder, M. C., Mikkola, H. K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Emergence+of+Hematopoietic+Stem+Cells+Is+Initiated+in+the+Placental+Vasculature+in+the+Absence+of+Circulation.">The Emergence of Hematopoietic Stem Cells Is Initiated in the Placental Vasculature in the Absence of Circulation.</a> Cell Stem Cell. 2, 252-263 (2008).</li></ol>

Bu protokol açıklanan deneysel prosedürleri plasental doku ve hematopoietik kök ve progenitör hücrelerin izolasyonu ve görüntüleme için izin verir. Fetal gelişim boyunca plasenta ve diğer fetal hematopoetik organlar HKH&#39;lerin beklenen hücresel verim ve sayısı hakkında kapsamlı bir özeti için Gekas, ark bakın. 2005. Plasenta gelişmekte HKH&#39;lerin ve diğer hematopoetik hücreler lokalizasyonu için Rodos ve ark bakın. 2008.

Malzeme Listesi: 100 mM Glukoz 10mM NaN3 15 ml konik tüpler (BD) 16-G, 18-G, 20-G, 22-G ve 25 G iğneler (BD) % 30 Sakkaroz Çözüm 35mm Petriler (BD) 4% Paraformaldehit 50 - m hücre filtreleri (Celltrics) 5 ml şırınga (BD) Alkalin Fosfataz Yüzey Takımı (Vektör Kırmızı, Vektör) Alkalin Fosfataz Yüzey Takımı III (Vektör Mavi, Vektör) Antibiyotik (Penisilin / Streptomisin, P / S) çözümü Kollajenaz tip I (Sigma) Cryomold Disposable Vinil Numune Kalıpları (10mm x 10mm x 5 mm, Doku-Tek) Sitokeratin İlköğretim Antikor (DakoCytomation) Diseksiyon forseps, paslanmaz çelik veya titanyum, sayı 5 veya 55 Drierite Fetal sığır serumu (FBS) Fisherbrand Plastik Mikroskop Slayt Mailer (Balıkçı) Glikoz oksidaz 1000u/ml Levamisole (Vector) Montaj medya, Sulu tabanlı Montaj Orta (VectamountTM AQ) Normal At Serumu (Vektör) Optimal Kesme Sıcaklığı (OCT, Doku-Tek) Peroksidaz Yüzey Takımı DAB (Vektör) Fosfat Tamponlu Salin (PBS) Kalsiyum / Magnezyum (w Ca2 + / Mg2 +) Fosfat Tamponlu Salin (PBS), Kalsiyum / Magnezyum (w / o Ca2 + / Mg2 +) olmadan Plastik Slayt Kutusu (100 slaytlar tutan) Plastik Boru (Nalgene 180 PVC Sigara toksik Otoklavlanabilir) Polipropilen Yuvarlak Tabanlı Tüp, 5 ml (12x75 mm, BD Falcon) Proteinaz K 20mg/ml Çözüm (Amresco) Araştırma Ürünler International Corp Süper Pap Kalem HT * Slayt işaretlerini (2,5 mm, Fisher) Doku kültürü U-şekilli (yuvarlak alt) tedavi 96-iyi ya da 24 kuyu levhalar (BD Falcon) Tween20, SigmaUltra (Sigma) Tyramide Amplifikasyon Kit (Invitrogen) Vectastain ABC Alkalin Fosfataz Standart Kiti (Vector) Vectastain ABC Standart Elite Kiti (Vector)

isolation and analysis of hematopoietic stem cells from the placenta

Hematopoetik kök hücreler (HKH&#39;lerin), kendi kendini yenileme ve bireyin ömrü boyunca kan soyların bütün hücre tipleri oluşturmak yeteneğine sahip. Tüm HKH&#39;lerin kendi havuzu boyutu kendini sürekli yenileyen hücre bölünmeleri tarafından yapılmaktadır, bundan sonra, embriyonik gelişim sırasında ortaya çıkar. HSC gelişme sürecini düzenleyen HKH&#39;lerin ve kritik gelişimsel olayların anatomik kökenli belirlenmesi, birçok anatomik siteler fetal hematopoez sırasında katılmak olarak karmaşık olmuştur. Son zamanlarda, büyük hematopoetik bir organ olarak HKH&#39;lerin (Gekas, ve ark 2005, Rodos, ve ark 2008) ve eşsiz microenvironmental nişler genişletilmiş plasenta belirledi. Sonuç olarak, plasenta, bunların ortaya çıkması ve ilk genişleme sırasında HKH&#39;lerin önemli bir kaynak. Bu makalede, biz E10.5 ve sırasıyla HKH&#39;lerin başlatılması ve plasenta HSC havuzu boyutu zirve gelişim evreleri ile ilgili E12.5 embriyolar, fare plasentanın izolasyonu için diseksiyon teknikleri gösteriyor. Buna ek olarak, flow sitometri veya fonksiyonel testleri kullanmak için tek hücre süspansiyonu içine plasental doku enzimatik ve mekanik ayrılma için optimize edilmiş bir protokol mevcut. Plasentanın tek hücre süspansiyonu için kollajenaz kullanımı HKH&#39;lerin yeterli verimi verdiğini bulduk. Plasenta HSC verimi etkileyen önemli bir faktör, önceden mekanik ayrılma derecesi, ve enzimatik tedavi süresi. Biz de onların hassas hücresel nişler içinde immünohistokimya HKH&#39;lerin gelişmekte olan görselleştirme için sabit-dondurulmuş plasental doku kesitlerinde hazırlanması için bir protokol sağlar. Hematopoetik spesifik antijenler parafin bölümlerinin hazırlanması sırasında korunur olarak, rutin plasental HKH&#39;lerin ve atalarıdır lokalize için sabit dondurulmuş bölümleri kullanabilirsiniz.

Watch this Scientific Journal Video about Isolation and Analysis of Hematopoietic Stem Cells from the Placenta at JoVE.com

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Biz, gelişimi sırasında büyük bir hematopoetik organ olarak plasenta tespit ettik. Biz, hematopoietik kök hücreler (HKH&#39;lerin) üretilen ve benzersiz microenvironmental nişler içinde plasenta genişletilmiş iki bulundu. Burada, fare plasenta HKH&#39;lerin izolasyonu ve görselleştirme için gerekli deneysel teknikler açıklanmaktadır.

Plasenta Hematopoetik Kök Hücre İzolasyonu ve Analizi

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

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A Hierarchical Stem-Cell System: Blood Cell Formation

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20.3K Görüntüleme.  University of California, Los Angeles. Biz, gelişimi sırasında büyük bir hematopoetik organ olarak plasenta tespit ettik. Biz, hematopoietik kök hücreler (HKH'lerin) üretilen ve benzersiz microenvironmental nişler içinde plasenta genişletilmiş iki bulundu. Burada, fare plasenta HKH'lerin izolasyonu ve görselleştirme için gerekli deneysel teknikler açıklanmaktadır.

Video: Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

İzolasyon ve Hematopoetik Kök Hücre Transplantasyonu (HKH&#39;lerin)

Biyoloji

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 ...

Isolation of Early Hematopoietic Stem Cells from Murine Yolk Sac and AGM

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These regions are crucial in establishing the blood system in the embryos and leads to the eventual movement of stem cells into the fetal liver and then development of adult stem cells in the bonemarrow. Early hematopoietic stem cells can be isolated from these organs through microdissection of the embryo followed by flow cytometric sorting to obtain a more pure population. It remains unclear how these stem cell populations contribute to the fetal and adult stem cell pool.   Also, our lab investigates how early stem cells functionally differ from fetal and adult hematopoietic stem cells. Furthermore, our lab sorts different populations of hematopoietic stem cells and test their functional role in the context of a variety of genetic models. In this video, we demonstrate the micro-dissection procedure we commonly use and also show the results of a typical FACS plotfter isolating these rare populations, it is possible to perform a variety of functional assays including: colony assays and bone marrow transplants. 



This video shows how to micro-dissect the yolk sac and aorta-gonad-mesonephros region from embryos and use flow cytometry to sort hematopoietic stem cells.

Murin Yolk Sac ve AGM Erken Hematopoetik Kök Hücre İzolasyonu

In the mouse embryo, early hematopoiesis occurs simultaneously in multiple organs, which includes the yolk sac and aorta-gonad-mesonephros region. These ...

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 ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

Lentiviral aracılı demonte sırasında Ex vivo İnsan Hematopoetik Kök Hücre Eritropoiesis

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Mürin Hematopoetik Kök Hücreler ve Lineage-kararlı progenitörlerin Fenotipik Analizi ve İzolasyon

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

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 ...

Retroviral Infection of Murine Embryonic Stem Cell Derived Embryoid Body Cells for Analysis of Hematopoietic Differentiation

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Manipulating temporal gene expression in differentiating embryonic stem cells (ESCs) can be achieved using inducible gene systems. However, generation of these cell lines is costly and time consuming. This protocol achieves rapid expression of a transgene in differentiating ES-derived cells and subsequent analysis of downstream hematopoietic differentiation.

Kemirgen Embriyonik retroviral Enfeksiyon Hematopoetik Farklılaşma Analizi için Hücre Türetilmiş embriyoidlerin Vücut Kök Hücreler

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for ...

Identification of Key Factors Regulating Self-renewal and Differentiation in EML Hematopoietic Precursor Cells by RNA-sequencing Analysis

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as leukemia and lymphoma. Elucidating the mechanisms controlling HSCs self-renewal and differentiation is important for application of HSCs for research and clinical uses. However, it is not possible to obtain large quantity of HSCs due to their inability to proliferate in vitro. To overcome this hurdle, we used a mouse bone marrow derived cell line, the EML (Erythroid, Myeloid, and Lymphocytic) cell line, as a model system for this study.
RNA-sequencing (RNA-Seq) has been increasingly used to replace microarray for gene expression studies. We report here a detailed method of using RNA-Seq technology to investigate the potential key factors in regulation of EML cell self-renewal and differentiation. The protocol provided in this paper is divided into three parts. The first part explains how to culture EML cells and separate Lin-CD34+ and Lin-CD34- cells. The second part of the protocol offers detailed procedures for total RNA preparation and the subsequent library construction for high-throughput sequencing. The last part describes the method for RNA-Seq data analysis and explains how to use the data to identify differentially expressed transcription factors between Lin-CD34+ and Lin-CD34- cells. The most significantly differentially expressed transcription factors were identified to be the potential key regulators controlling EML cell self-renewal and differentiation. In the discussion section of this paper, we highlight the key steps for successful performance of this experiment.
In summary, this paper offers a method of using RNA-Seq technology to identify potential regulators of self-renewal and differentiation in EML cells. The key factors identified are subjected to downstream functional analysis in vitro and in vivo.

RNA-sequencing and bioinformatics analyses were used to identify significantly and differentially expressed transcription factors in Lin-CD34+ and Lin-CD34- subpopulations of mouse EMLcells. These transcription factors might play important roles in determining the switch between self-renewing Lin-CD34+ and partially differentiated Lin-CD34- cells.

RNA dizileme Analizi ile EML Hematopoetik Öncü Hücreler Self-yenileme ve farklılaşma Düzenlenmesi Anahtar Faktörler belirlenmesi

Hematopoietic stem cells (HSCs) are used clinically for transplantation treatment to rebuild a patient&#39;s hematopoietic system in many diseases such as ...

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 ...