Yazarlar, güncel film ve slayt düzenleme hakkında yardım için Sayın Kyle Holden ve Sayın Jonathan Lucas için hücre izolasyonu, teknik yardım, Bayan Haiying Pan ve Bay Ian Bellayr büyük takdir. Dr: Geçtiğimiz yıllarda modifiye preplate tekniklerinin geliştirilmesi ile ilgili olan kişilerin aşağıdaki listeye çok teşekkürler. Burhan Gharaibeh, Baohong Cao, Deasy Bridget, Thomas R. Payne, Aiping Lu, Hairong Peng, Hideki Oshima, Bo Zeng, Xiaodong Mu, Kimimasa Tobita Ron Jankowski, Makato Ikezawa. James H. Cummins, Ryan Pruchnic, Joseph Feduska, Rebecca C. Schugar, Haiying Pan ve Jessica Tebbets teknik yardım için teşekkür ediyoruz. Yazar ayrıca Müsküler Distrofi Derneği (MDA), Ulusal Sağlık Enstitüleri (NIH), Savunma Bakanlığı (DOD), UPMC, Pittsburgh Çocuk Hastanesi, Anabilim Dalı Bu çalışma için mali destek kabul etmek gibi. Ortopedi ve Travmatoloji ve Pittsburgh Üniversitesi.

Genel olarak, komple değiştirilmiş preplate prosedürü hazırlık 1-2 gün, 1 hafta teknik yordamı gerçekleştirmeden, 2-3 gün hücre immünsitokimya kimlik, kök hücre nüfus büyümesi ek bir 2-3 hafta gerektirir. Kök hücre kültürü için gerekli ekipman, bir tezgah üstü santrifüj, CO2 inkübatör, Laminer akış doku kültürü kaputu ve floresans ve bir dijital kamera ile donatılmış bir inverted mikroskop içeren diğer hücre kültür sistemleri bu benzer . Izole edilen kök hücreler de klonlanmış olabilir ve mevcut ya da gelecekteki çalışmaları için 1,2 sıvı nitrojen içinde uzun vadeli saklanabilir. Bu prosedür için kullanılan tüm reaktifler ve malzemelerin steril olması ve aseptik olarak ele gerekir. Adım 1: Hazırlık <ol><li> Kollajen kat doku kültürü yemekleri ve balonları: buzağı deri (1 litre suda 0.1 g; Sigma-Aldrich) türetilen Tip I kollajen. Kat için plasticware içerir: T-25 şişeler 1,2 önce açıklandığı gibi, 6 veya 12 sıra tabak, yemekler (60 ve 100 mm, BD Falcon). Yavaşça kollajen bile kaplama sağlamak için 4 saat boyunca plasticware, her yarım saatte bir sallamak. Kollajen çözüm daha sonra kaldırılır. Ve kaplı plasticware sonra kısırlık sağlamak için, UV ışık ve kaput, hem doku kültürü kaputu kurumaya izin verilir, ve son olarak Tekrar kapatılan veya daha sonra kullanmak için kapalı. </li><li> Hücre kültür ortamı hazırlayın, 50ml Isı İnaktif Fetal Bovine Serum (FBS, Invitrogen), 50ml At Serum (HS, Invitrogen) ve 5 ml Civciv Embriyo Extract (CEE; Doğru Chemical Co 400ml DMEM (Invitrogen DMEM, yüksek glukoz): ) bir tek 0.22 mikron 500 ml steril filtre sistemi (Corning) kullanarak filtreli elektrik süpürgesi olacak. Steril penisilin / streptomisin çözüm (Invitrogen) sonunda 100 Birimler / mL eklenecektir. Hazırlanan çözümler en az bir ay süreyle 4 ° C&#39;de muhafaza edilebilir. Bu çözüm, balon hücreleri ayırmak için kullanılan bölme kültürleri veya transplantasyon için hücrelerin hazırlanması. </li><li> Hücre izolasyon çözümleri kadar Isınma: Aşağıdaki reaktifler 37 kadar ısınmış olması için ° C hücre izolasyonu için kullanılması önce: Hank Tamponlu Tuz Çözüm (HBSS, Invitrogen); 0.2% (ağırlık / hacim) kollajenaz-tip XI (Sigma Dispase 2.4 units/1mL (Invitrogen);% 0.5 &#39;lik (ağırlık / hacim) tripsin (Invitrogen) başına 3.200 ml HBSS kollajen sindirim birimlerinin ortalama-Aldrich). </li><li> Cerrahi teçhizat hazırlık sterilize:: Cerrahi işlem de dahil olmak üzere steril cerrahi aletler gerektiren farklı büyüklükteki forseps, mikro-makas, iris makası ve / veya bisturi ağızları. Ayrıca, 15 ve 50 ml polipropilen santrifüj tüpleri (BD Falcon); Hücre süzgeç (gözenek büyüklüğü 70 m) (BD Falcon), 18 - 23 - ve 27-gauge iğne (BD PrecisionGlide), 10 - veya 20 ml steril şırıngalar (BD). </li></ol> Adım 2: Doku biyopsi, izolasyon ve mekanik disosiasyon 1-4 Doku biyopsileri steril bir kültür kaputun altında yapılır ve taze hasat tendon ve iskelet kasları, wild-tip farelerden hindlimb (Kadın C57BL/6J, 4-5 yaş hafta ya da daha genç, Jackson tibia veya soleus kas elde edilen Laboratuvar). Biyopsi örnekleri görünür bir bağ dokusu, kan damarları ve yağ dokusu daha sonra kaldırılır. Tendon ve kas dokuları, deri ve kemik kalıntıları kaldırmak için bir cerrahi diseksiyon mikroskobu altında dikkatle tespit ve bir tabak içinde bulunduğu soğuk (4 ° C) HBSS (% 5 FBS ile takviye). Izole dokularda sonra soğuk HBSS içeren kollajen kaplı yemekleri ayrı ayrı yerleştirilir ve kaba bir bulamaç haline mikro-makas ve / veya bisturi ağızları kullanarak (&lt;1x1 mm 3) kıyılmış olacak . Adım 3: Enzimatik sindirim dokuların: Kıyılmış doku adımları 2,4,5 bir dizi enzimatik ayrışmış <ol><li> Ayrı 15 ml tüpler ve 4 ~ 3500 rpm&#39;de santrifüj ° C 5 dakika içine kıyılmış doku çamurlar aktarın. </li><li> Süpernatantı HBSS ile yıkayın ve tekrar santrifüj tekrarlayın. </li><li> Süpernatantı prewarmed% 0.2 kollajenaz tip XI (Sigma) 10 ml ekleyerek bu çamurlar sindiremez. 60 dakika için 37 ° ° C&#39;de sürekli tüpler hafifçe sallanan. Alternatif olarak, her 10 dakikada bir, el sallayın. </li><li> Santrifüj ve 10 ml dispase (2.4 Birimler / mL Gibco) çözümü bu çamurlar tekrar süspansiyon. Elleriyle sallayarak veya her 10 dakikada bir hafifçe sallanan 37 ° C&#39;de 45 dakika inkübe edin. </li><li> 10 ml% 0.2 tripsin HBSS çözüm bu çamurlar Santrifüj ve yeniden askıya. Kuluçka tek hücreleri, mikroskop altında süspansiyon gözlemleyerek yapılabilir dokulardan yayımlanan hangi ölçüde değişecektir. Tüpleri ile tersine ° C 37 30 dakika aşmak için tavsiye veya her 10 dakikada bir nazikçe sallanan değildir. </li><li> Sonuçlanan hücre Santrifüjs ve yeniden askıya hücre pelletini Silahların Yayılmasını Önleme Orta (PM) 10 ml. </li><li> Iğne bir dizi özü geçerek hücre süspansiyonu ayrıştırmaları. Hücreler daha sonra bir 18G ile 23G 2 kez geçti ve nihayet bir 27G iğne. </li><li> 70 mikron hücre süzgeç vasıtasıyla hücre özü iletin. </li></ol> Adım 4: hücre adezyon oranı 1-3 dayalı farklı hücre popülasyonlarının izole Preplating <ol><li> Santrifüj ve hücre pelletleri tekrar süspansiyon Silahların Yayılmasının Önlenmesi Orta. </li><li> Plate hücre kollajen kaplı bir T-25 balonuna (veya 60 mm yemek) üzerine karışımları ve PP1 olarak işaretlemek. Hücre süspansiyonu, mikroskop altında çok sayıda kırılma çekirdek ve diğer enkaz sahip olarak dikkat edilmelidir. </li><li> 37 inkübe ° C, 2 saat boyunca nemlendirilmiş bir,% 5 CO 2 inkübatör . </li><li> T-25 balonuna (veya 60 mm çanak) ve mark PP2 olarak yeni bir kollajen kaplı nonadherent hücreleri aktarın. PP1 etiketli plakasına PM 5 ml ekleyin. Balona ekleyebilirsiniz erken yapışan hücreler çoğunlukla miyofibroblastlar 3. </li><li> (Ya da çanak) T-25 balon yeni bir kollajen kaplı nonadherent hücreleri transferi ve PP3 olarak işaretlemek, başka bir 24 saat süreyle inkübatör şişeler dönün. Silahların Yayılmasının Önlenmesi Orta PP2 etiketli plakasına 5 ml ekleyin. Bu balona eklemek Bu kalarak hücreleri çoğunlukla fibroblastlar 2,3. </li><li> Inkübatör şişeler dönün ve 24 saat içinde nüfus PP6 oluşturuluncaya kadar işlemi tekrarlayın. Her grup askıya hücreleri korumak ve çoğalmaya başlar ve ters bir mikroskop kullanılarak düzenli olarak incelemeye olanak sağlar. Kas uydu hücreleri genellikle PP5 2 çoğunlukla görülürken PP3-PP4 çoğunlukla matür. </li><li> Yavaş yavaş kalarak hücrelerinin çoğu genellikle PP6 takın. Bu aşamada hücreler küçük, yuvarlak, ışık kırılma görünür ve sayısı çok azdır ve bir kök hücre nüfus 1,2 olarak kabul edilir. </li></ol> Adım 5: Tanımlama ve izole edilen kök hücrelerin genişlemesi <ol><li> Izole PP6 hücreleri sayıca çok azdır ve günlük değiştirildi FBS zengin Silahların Yayılmasını Önleme Orta (% 20 DMEM içinde FBS) muhafaza edilmesi gerekir. Hücrelerin çoğu, kültür, aşağıdaki 1-2 hafta boyunca PP6 kültür kalıp mevcut, ama büyük, sağlıklı PP6 nüfus genellikle genişleme ek bir 1-2 hafta sonra oluşturulur. Farelerden elde edilen izole edilen kök hücreler, son derece 1 (SCA1) kök hücre antijen, CD34, fetal karaciğer kinaz 1 (Flk1) ve immünositokimyasal boyama aracılığı ile görsel olarak diğer kök belirteçlerinin 1,2,6,7 ifade eder. Bu kök hücrelerin, aynı zamanda birden fazla farklılaşma kapasiteleri 3,6,8 sergilemek . </li><li> Izole hücreler korunur ve farklılaşma 1,2 önlemek için (iyi ya da &lt;% 20-30 izdiham şişesi veya çanak 12 50-100 hücreleri iyi tabaklar /) düşük bir kültür hücre yoğunluğu genişletilmiş. Çok az sayıda hücre genişleme sırasında klonlar formu ve bu klonlanmış kök hücrelerinin başka herhangi bir yeterlilik çalışmaları için uzun süre proliferasyonu (LTP) yaptırmalıdırlar. Klonlanmış kök hücreler de% 10 DMSO ile PM orta örneğin genel hücre depolama prosedürleri kullanarak sıvı azot deposu olabilir. Kök hücreler, herhangi bir diğer geri çalışmalar için düşük geçiş stoklarının bir dizi olun. </li></ol>

<ol>
	<li>Gharaibeh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+slowly+adhering+cell+fraction+containing+stem+cells+from+murine+skeletal+muscle+by+the+preplate+technique.">Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique.</a> Nat Protoc. 3, 1501-1509 (2008).</li><li>Qu-Petersen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+population+of+muscle+stem+cells+in+mice:+potential+for+muscle+regeneration.">Identification of a novel population of muscle stem cells in mice: potential for muscle regeneration.</a> J Cell Biol. 157, 851-864 (2002).</li><li>Li, Y. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+muscle-derived+cells+into+myofibroblasts+in+injured+skeletal+muscle.">Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle.</a> Am J Pathol. 161, 895-907 (2002).</li><li>Watt, D. J., Lambert, K., Morgan, J. E., Partridge, T. A. &. a. m. p. ;. a. m. p., Sloper, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+donor+muscle+precursor+cells+into+an+area+of+muscle+regeneration+in+the+host+mouse.">Incorporation of donor muscle precursor cells into an area of muscle regeneration in the host mouse.</a> J Neurol Sci. 57, 319-331 (1982).</li><li>Lu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+myogenic+progenitor+populations+from+Pax7-deficient+skeletal+muscle+based+on+adhesion+characteristics.">Isolation of myogenic progenitor populations from Pax7-deficient skeletal muscle based on adhesion characteristics.</a> Gene Ther. 15, 1116-1125 (2008).</li><li>Lee, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+isolation+of+muscle-derived+cells+capable+of+enhancing+muscle+regeneration+and+bone+healing.">Clonal isolation of muscle-derived cells capable of enhancing muscle regeneration and bone healing.</a> J Cell Biol. 150, 1085-1100 (2000).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+receptors+in+the+maturation-dependent+adenoviral+transduction+of+myofibers.">The role of receptors in the maturation-dependent adenoviral transduction of myofibers.</a> Gene Ther. 8, 627-637 (2001).</li><li>Cao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+stem+cells+differentiate+into+haematopoietic+lineages+but+retain+myogenic+potential.">Muscle stem cells differentiate into haematopoietic lineages but retain myogenic potential.</a> Nat Cell Biol. 5, 640-646 (2003).</li><li>Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+based+on+muscle+stem+cells.">Regenerative medicine based on muscle stem cells.</a> J Musculoskelet Neuronal Interact. 8, (2008).</li><li>Gates, C. B., Karthikeyan, T., Fu, F. &. a. m. p. ;. a. m. p., Huard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regenerative+medicine+for+the+musculoskeletal+system+based+on+muscle-derived+stem+cells.">Regenerative medicine for the musculoskeletal system based on muscle-derived stem cells.</a> J Am Acad Orthop Surg. 16, 68-76 (2008).</li></ol>

Bu, bazı yetişkin dokularda çok kök hücreler içerdiğini kabul edilmiştir. Çalışmanın son birkaç yıl içinde, kök hücre, iskelet kas ve tendon gibi yumuşak kas-iskelet dokuları, tespit edilmiştir. Tendon ve iskelet kası yetişkin kök hücreleri izole etmek için başarılı bir laboratuvarda kullanılan olmuştur değiştirilmiş preplate tekniği olarak bilinen bu akım protokol detaylarının bir prosedür. Hücreleri yukarıda açıklanan değiştirilmiş preplate tekniği kullanarak çeşitli pop...

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isolating stem cells from soft musculoskeletal tissues

Yetişkin kök hücreleri uzun rejeneratif tıp uygulama ile ilgili olarak ele alınmıştır. Yetişkin kök hücreleri, çok soyundan hücre farklılaşması ve kendi kendini yenileme yeteneği geçmesi etkileyici yetenekleri nedeniyle yaralı ve hastalıklı dokuların tedavisi için büyük bir heyecan yarattı. En önemlisi, bu niteliklere otolog hücre nakli tedavileri kullanmak için onları avantajlı hale getirmiştir. Geçerli protokol, kas-iskelet sistemi, örneğin, tendon ve kas, yumuşak doku 1 değiştirilmiş preplate tekniğini okuyucularına tanıtacak<sup&gt; St</sup&gt; Orta kollajen kaplı şişelerde yerleştirilen enzimatik olarak ayrışmış ve sonra. Orta ve kalan yüzen hücreler oluşur süpernatant, seri, yeni şişeler için günlük transfer edilir. Kök hücreler (seri transferleri veya preplates) genellikle 5-7 gün sürer şişeler uymak için en yavaş. Bu tekniği kullanarak, yetişkin kök hücreler bu dokuların mevcut oldukça non-invaziv prosedürler yoluyla kolayca hasat edilebilir.

Watch this Scientific Journal Video about Isolating Stem Cells from Soft Musculoskeletal Tissues at JoVE.com

Isolating Stem Cells from Soft Musculoskeletal Tissues

Balona hücre bağlılık hızına göre kas-iskelet yumuşak dokulardan Yalıtımlı yetişkin kök hücreleri.

Yumuşak Kas-iskelet Dokular Kök Hücre Yalıtımlı

Adult stem cells have long been discussed in regards to their application in regenerative medicine. Adult stem cells have generated a great deal of ...

isolating-stem-cells-from-soft-musculoskeletal-tissues

Research

JoVE Journal

Biology

13.3K Görüntüleme.  Stem Cell Research Center, Childrens Hospital of Pittsburgh of UPMC. Balona hücre bağlılık hızına göre kas-iskelet yumuşak dokulardan Yalıtımlı yetişkin kök hücreleri.

Video: Isolating Stem Cells from Soft Musculoskeletal Tissues

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

İnsan Embriyonik Kök Hücre Kromatin Immunoprecipitation

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Derivation of Mouse Trophoblast Stem Cells from Blastocysts

Specification of the trophectoderm is one of the earliest differentiation events of mammalian development. The trophoblast lineage derived from the trophectoderm mediates implantation and generates the fetal part of the placenta. As a result, the development of this lineage is essential for embryo survival. Derivation of trophoblast stem (TS) cells from mouse blastocysts was first described by Tanaka et al. 1998. The ability of TS cells to preserve the trophoblast specific property and their expression of stage- and cell type-specific markers after proper stimulation provides a valuable model system to investigate trophoblast lineage development whereby recapitulating early placentation events. Furthermore, trophoblast cells are one of the few somatic cell types undergoing natural genome amplification. Although the molecular pathways underlying trophoblast polyploidization have begun to unravel, the physiological role and advantage of trophoblast genome amplification remains largely elusive. The development of diploid stem cells into polyploid trophoblast cells in culture makes this ex vivo system an excellent tool for elucidating the regulatory mechanism of genome replication and instability in health and disease. Here we describe a protocol based on previous reports with modification published in Chiu et al. 2008.

In this video, we demonstrate the isolation of mouse blastocysts and the derivation of trophoblast stem cells from blastocysts. We also describe conditions for maintenance of the stem cell property as well as induction of differentiation in culture.

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 ...

Isolation of Stem Cells from Human Pancreatic Cancer Xenografts

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny1. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24low/-)2. Since then, CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties, including aldehyde dehydrogenase (ALDH) activity, have also been used to isolate CSCs from malignant tissues3-5.
Recently, we and others identified CSCs from pancreatic adenocarcinoma based on
ALDH activity and the expression of the cell surface antigens CD44 and CD24, and CD1336-8. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic, but ALDH+ cells are relatively more invasive8. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human
xenografts9. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,
a fluorescent substrate of ALDH10. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells.

Cancer stem cells (CSCs) have been identified in a number of malignancies. In this protocol we describe a flow cytometric method utilizing aldehyde dehydrogenase activity and CD44 and CD24 expression to isolate CSCs from human pancreatic adenocarcinoma xenografts. These viable cells can then be used in functional and analytical studies.

İnsan Pankreas Kanseri Xenografts Kök Hücre İzolasyonu

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal ...

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 ...

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

İnsan İsteyerek Pluripotent Kök Hücre seçme ve ayırma Kolonileri Yetişkin Fibroblastlar gelen reprogrammed

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

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 ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

Fare Kalpten İlköğretim Melanocyte gibi Hücreleri Ayırma

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

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 ...

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly in tissues that have a high turnover rate such as the intestinal epithelium. However, adult stem cells are also subject to aging processes and the concomitant decline in function. The Drosophila midgut has emerged as an ideal model system to study molecular mechanisms that interfere with the intestinal stem cells&#8217; (ISCs) ability to function in tissue homeostasis. Although adult ISCs can be easily identified and isolated from midguts of young flies, it has been a major challenge to study endogenous molecular changes of ISCs during aging. This is due to the lack of a combination of molecular markers suitable to isolate ISCs from aged intestines. Here we propose a method that allows for successful dissociation of midgut tissue into living cells that can subsequently be separated into distinct populations by FACS. By using dissociated cells from the esg-Gal4, UAS-GFP fly line, in which both ISCs and the enteroblast (EB) progenitor cells express GFP, two populations of cells are distinguished based on different GFP intensities. These differences in GFP expression correlate with differences in cell size and granularity and represent enriched populations of ISCs and EBs. Intriguingly, the two GFP-positive cell populations remain distinctly separated during aging, presenting a novel technique for identifying and isolating cell populations enriched for either ISCs or EBs at any time point during aging. The further analysis, for example transcriptome analysis, of these particular cell populations at various time points during aging is now possible and this will facilitate the examination of endogenous molecular changes that occur in these cells during aging.

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.

Yetişkin gelen Bağırsak Kök Hücreler Yalıtımlı<em&gt; Drosophila</em&gt; FACS Midguts Yaşlanma sırasında Kök Hücre Davranışı Eğitim için

Aging tissue is characterized by a continuous decline in functional ability. Adult stem cells are crucial in maintaining tissue homeostasis particularly ...