Dr Lucy Senter, Dr. Brigit Willeford ve MSU ALAC akredite fare tesisi, eğitim ve hayvan bakımı ile ilgili yardım için Mike Basett; Dr. Dwayne Wise Dr Neal Birinci ve yazının düzenleme için Kourtney Wilkinson: Yazarlar, paha biçilmez bir yardım kabul etmek istiyorum Cesar Monroy Hannah Swoope ve Bobbie Huddleston MSU video üretimi ile yardım için mikroskopi ve görüntü yakalama ile yaptığı yardım için. Bu araştırma, Mississippi State Üniversitesi Kurumsal Araştırma ve Biyolojik Bilimler Bölümü Ofisi tarafından finanse edildi.

Bölüm 1: Hamile Fare Laparotomi <ol><li> Servikal dislokasyon kullanarak, 10,5-11,5 DPC, hamile bir C57BL6J kadın fare euthanize. </li><li> Karın antimikrobiyal sabun ile temizleyin ve sonra, tıraş. </li><li> Tıraştan sonra karın, bir tuzlu su çözeltisi ile yıkayın. </li><li> Sonra steril bir gazlı bez ile karın kurulayın. </li><li> Yeni bir steril alan fare ile kaplayın. </li><li> Forseps ve diseksiyon makas kullanarak ventral kesi olun. </li><li> Belirlenmesi ve karın boşluğuna kadar tüm rahim kaldırmak. Fare embriyoları rahim içinde görünür olacaktır. </li><li> D-PBS ile dolu bir petri içine rahim transferi ve buz üzerinde tutmak. </li><li> Steril bir bisturi ve forseps kullanarak, plasenta ve her embriyo ekstra embriyonik dokularda çıkarın. </li><li> D-PBS ile dolu yeni bir petri içine embriyo transferine. </li><li> Fare embriyo uzunluğunu ölçün. Biz boyutları embriyonun yaşına göre farklılık bulundu. 6mm ortalama ölçülen DPC örneğin 8.5, 10.5 DPC 11mm ortalama ölçülen ve 16 mm ortalama 12.5 DPC ölçülür. </li><li> Daha sonra, DNA ekstraksiyonu için kuyrukları kaldırmak. </li></ol> Bölüm 2: Gonadal Ridge Diseksiyon Stereomikroskopta ve Işık kaynağı Schott Fostec altında <ol><li> Petri kabındaki bir filtre kağıdı yerleştirin. Sonra, embriyo kuru ve diseksiyon için hareketsiz fare embriyo filtre kağıdının üstüne koyun. </li><li> Steril bir bistüri kullanarak, fare embriyo göbek kordonu kökeni üzerinde enine kesilmiş olun. </li><li> Stereomikroskop altında, steril ince forseps ve steril bir ince alay iğne kullanarak, üriner sistem görünür olduğunu bu nedenle bağırsak ve karaciğer kaldırmak. Böbrekler yanal karın boşluğunda bulunan ve mesane, böbrekler göre medial ve kaudal bulunmaktadır. Gonadal sırtlar mesane iki tarafında bulunması nedeniyle embriyonun erkek olarak tespit edilebilir. Kadın, gonadal sırtlar böbrekler kaudal lateral sonuna kadar sıkıca bağlıdırlar. </li><li> Arkasında bir iğne kaydırarak gonadal sırt dışarı Peel. Gonadal sırtı destekleyen dokuların kesim tarafından kaldırılır. </li><li> Gonadal sırtlar taze D-PBS ile dolu yeni bir petri kabına aktarın. Mikroskopik, erkek gonadal sırt, büyük ve oval soyulmuş olmalıdır. Buna karşılık, dişi gonadal sırt fark olmalıdır, uzatılmış şekli ve erkek gonadal sırt kıyasla daha küçük olabilir. </li></ol> Bölüm 3: Ridge Sindirim Gonadal Altında stereomikroskopta: <ol><li> Mesonephros Diseksiyon: ince ve keskin bir iğne kullanılarak mesonephros ve sırt gonad ayırın. <ul><li> PGC türetme süreç içinde büyümek bir somatik doku olması nedeniyle gonadal sırt mesonephros kaldırmak için önemlidir. </li></ul></li><li> Gonadal Ridge Sindirim: taze D-PBS 0.5ul açılan temiz gonadal sırtlar toplayın. Kollajenaz / Dispase Çözüm 20 ml (1mg/ml Final konsantrasyon) ekleyin. <ul><li> Embriyolar ayrı ayrı işleme tabi tutulmalıdır. Her embriyo kendi ayrı ve etiketli petri olmalıdır. </li></ul></li><li> Gonadal Ridge kıyma: gonadal sırt, steril bir iğne ve steril ince forseps No 4 kullanarak küçük parçalar halinde kesin. </li><li> Kuluçka: Petri kapları 37 bir kuluçka aktarılır ° C de 15 dakika boyunca. <ul><li> Inkübasyon süresi dokular arasında değişebilir. </li></ul></li><li> Pipetleme: 15 dakika sonra, doku pipetleme ayrışmış olabilir. 40 -100 mm çapları arasında çekilmiş cam kapiller pipetler kullanarak, yukarı ve aşağı pipetleme tarafından parçaları kadar Break. Bu küçük öbekler şeklinde olacaktır. Nihai bir yıkama için bir ön ısındı D-PBS damla 0.5 ml yığınları aktarın. </li><li> Yıkadıktan sonra yığınları, Eppendorf tüplerine transfer. <ul><li> Doku üzerinde kollajenaz maruz bırakmayın. </li><li> Tek bir hücre süspansiyonu yapmayın. Bu koloni oluşumu için önemlidir. Tek bir hücre süspansiyonu ise, PGCs farklılaştırmak ve göç eğilimindedir. </li><li> Bu işlemi 20 dakika daha uzun olmamalı, ya da hücreler canlılığını kaybedecektir. </li></ul></li><li> Oda sıcaklığında 5 dakika 2000 rpm&#39;de santrifüjleyin. </li><li> Santrifüj sonra, süpernatant kaldırmak ve medya dışında ilave vuruş (37 ° C&#39;de Önceden ısıtılmış) 0.5 ml pelet tekrar süspansiyon. </li></ol> Bölüm 4: İlkel Germinal Hücreler Kültür &quot;Sen, izolasyon ve derivasyon PGCs canlılığını korumak için hızla bir sonraki adımları gerçekleştirmeniz gerekir. <ol><li> Bu süreç 24 saat önce Zhang, J. ve ark 2008 protokolü takip mitotik olarak inaktive MEF-CF1 besleyici tabaka hazırlamalıdır. <ul><li> Bir pregna 20 yemekleri hazırlayınnt dişi fare (6-8 embriyoların). </li><li> MEFS 5-6 pasajlar sonra (büyümeyi teşvik etmek ve önlemek farklılaşma) besleyici tabaka yeteneğini kaybedersiniz. </li><li> 48 saatten daha büyük MEFS kullanmayın. </li></ul></li><li> Kaplama PGCs hemen önce, MEF orta yavaş yavaş MEF besleyici tabaka kaldırılmış olmalıdır. </li><li> Daha sonra, MEF besleyici tabaka üzerine, önceden ısıtılmış takviye nakavt orta / iyi 0,5 ml ekleyin. <ul><li> MEF medya PGCs, farklılaşma indükler fetal sığır serumu içerir. </li><li> Nakavt orta specie özel büyüme faktörleri ile takviye edilmelidir hemen önce büyümeye faktörünün etkinliği sağlamak için kullanmak. </li></ul></li><li> MEF besleyici tabaka üzerinde simetrik dağılımı, plaka PGCs kullanma. <ul><li> Bu parçaları çok yakın iseler, agrega ve kötü eklemek veya ayırt başlamak yoğun koloniler yapmak eğilimindedir. </li><li> Kültür yemekler Embriyonik Germinal hücre hattı adı, geçiş numarası ve tarih ile etiketlenmiş olmalıdır. </li></ul></li><li> Dikkatlice bir inkübatör içine 37 ° C ve% 5 CO 2 kültür kaplarına aktarın. </li><li> Hücreler her 24 saatte bir izleyin. </li><li> 48 saat sonra, koloni eki rahatsız etmemek için 250 ml medya kültürünün en üst çıkarın. Daha sonra, 250 ml taze takviye knock-out medya (37 ° C&#39;de önceden ısıtılmış) ekleyin. </li><li> 7 adım sonra, başka bir 48 saat bekleyin ve sonra, tamamen kaldırmak ve tüm medya taze knock-out takviye medya (37 ° C&#39;de önceden ısıtılmış) ile değiştirin. PGC koloni oluştururlar kadar 8-10 gün boyunca bu 2 günde yapın. </li></ol> Bölüm 5: Embriyonik Germinal Hücreler <ol><li> PGC izolasyon 10 gün sonra, embriyonik germinal hücre kolonileri oluşur. </li><li> Embriyonik germinal hücre hatları manuel geçişleri her 8-10 gün canlı tutulur. Farklılaşmamış bir morfoloji sunmak ve pluripotency belirteçlerinin alkalyne fosfataz, Ekim-4, SSEA-1, ve SOX-2 gibi ifade etmeye devam </li></ol> Notlar <ul><li> Steril / aseptik koşullarda kültür odası gereklidir. </li><li> Türetme ve kültür için, PGCs Arıtma Sınıf II Bio Güvenlik Kabinesi işlenir. </li><li> Inkübatörü nemlendirilmiş bir 37 ° C ve% 5 CO 2 inkübatör. </li><li> Tüm medya ve reaktifler 4 kayıtlı 0,2 um filtre ° C kullanmak için önce filtre ve kullanmadan önce 37 ° C önceden ısıtılmış. </li><li> Diseksiyonu adımları sırasında, tüm embriyolar ve dokuların buz üzerinde tutulmalıdır. </li><li> Tüm reaktif şişeler, bir kabin içine yerleştirmeden önce etanol ile dekontamine. </li><li> Eldiven, laboratuvar önlüğü ve hemşire kapaklar kullanılması zorunludur. </li></ul> Sonuç Biz izole etmek için nasıl bir video türetmek ve gonadal sırtlar 10,5-11,5 DPC fare embriyo kültür embriyonik germinal hücrelerin sağladı. Embriyonik germinal hücre hatları tekrarlanabilir izolasyon ve uzun vadeli kültürü, erken embriyonik gelişim, genetik ve epigenetik germinal desenleri, gonad oluşumu çalışma için kritik bir temel sağlar, erkek ve dişi embriyolarda gametogenezis gelişim sürecinde organları çevreleyen çevresel etkileri, ve kanser ve kısırlığa neden yollarının belirlenmesi. <img src="/files/ftp_upload/1635/1635figure1.jpg" alt="Şekil 1" />

<ol>
	<li>DeFelici, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell Biology: A laboratory Handbook. 1, 73-85 (1998).</li><li>Donovan, P. J., De Miguel, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+Germ+Cells+into+Stem+Cells.">Turning Germ Cells into Stem Cells.</a> Current Opinion in Genetics and Development. 13, 463-471 (2003).</li><li>Labosky, P. A., Hogan, B. L. M., Sharp, P. T., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Part+12:+Mouse+Primordial+Germ+Cells:+isolation+and+in+vitro+culture.">Part 12: Mouse Primordial Germ Cells: isolation and in vitro culture.</a> Molecular embryology: methods and protocols. 97, (1999).</li><li>Mc Laren, A., Southee, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+Mouse+Embryonic+Germ+Cells+into+Meiosis.">Entry of Mouse Embryonic Germ Cells into Meiosis.</a> Developmental Biology. 187, 107-113 (1997).</li><li>Yoshimizu, T., Obinata, M., Matsui, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-specific+tissue+and+cell+interactions+play+key+roles+in+mouse+germ+cell+specification.">Stage-specific tissue and cell interactions play key roles in mouse germ cell specification.</a> Development. 128, 481-490 (2001).</li><li>Zhang, J., Hhvorostov, I., Teitell, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+MEFs+to+Matrigel+I:+Passaging+hESCs+in+the+Presence+of+MEFs.">From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs.</a> J Vis Exp. , (2008).</li></ol>

Mississippi State Üniversitesi: Bu araştırma, inceleme ve Kurumsal Hayvan Bakım ve fare protokol # 08030 (Reaktif Oksijen germ line gelişimi ve tümör oluşumu Türlerin-ROS Rolü protokol Holding ve üreme) Kullanımı Komitesi (IACUC) onayı sonrası gerçekleştirildi.

Forseps No 14 ve 15, neşter 35 mm, diseksiyon makas, cerrahi alanlar, antimikrobiyal sabun, tuzlu solüsyon% 0.9, gazlı bez, buz köpüğü konteyner, jilet, bistüri 35 mm, ince forseps 4 ve 5 sayılı, ince alay iğne ile kağıt yaprak sapı, filtre ve cam pipetler çekti.<br /<br /&gt; Ekipman: diseksiyon stereomikroskopta, ışık kaynağı Schott Fostec, inverted mikroskop, 10 mikropipet ve 200ul, santrifüj, biyo güvenlik Dolabı.

isolation and derivation of mouse embryonic germinal cells

Embriyonik germinal hücrelerin yeteneği (EG), kanser ve kısırlık hakkındaki hipotezi adresi için mükemmel bir model, erken gelişim aşamalarında, ilkel germinal hücreleri (PGCs) içine ve daha sonra gamet içine ayırt etmek için. Bu protokol, 10,5-11,5 gün coitum (DPC) fare embriyoları, gelişmekte olan gonadlar ilkel germinal hücreleri izole etmek için nasıl gösterir. Fare embriyoları gonadal sırtlar Geliştirme (C57BL6J) sonra daha önce mitotik nakavt medya huzurunda mitomisin C ile inaktive edilmiş ve Lösemi İnhibitörü ile desteklenmiş bir fare embriyo fibroblast besleyici tabaka (MEF-CF1) kaplama kollajenaz ile mekanik parçalanması, ayrışmış edildi Faktörü (LIF), temel Fibroblast Büyüme Faktörü (bFGF) ve Kök Hücre Faktörü (SCF). PCG kimlik, kültür koşulları, izolasyon, ve kurulması için optimize edilmiş, bu yöntemleri kullanarak, en fazla 40 gün için EG hücreleri uzun vadeli kültürleri izin verir. Embriyonik germinal hücre hatları, pluripotent devletin yaygın olarak kullanılan belirteçlerin embriyonik fenotip ve ifade gösterdi. İzolasyon ve kültür germinal hücrelerin türetme gelişimini anlamak ve in vitro genetik ve epigenetik düzeyde oksidatif strese maruz kaldıktan sonra birikimli hasar izlemek için bir fırsat sunmak için bir araç sağlar.

Watch this Scientific Journal Video about Isolation and Derivation of Mouse Embryonic Germinal Cells at JoVE.com

Isolation and Derivation of Mouse Embryonic Germinal Cells

Ilkel germinal hücrelerin ayırt etmek için erken geliştirme aşamasında, embriyonik germinal hücreler, kanser ve kısırlık hakkındaki hipotezi adresine yeteneği mükemmel bir model. Bu protokol, 10,5-11,5 gün coitum fare embriyoları, gelişmekte olan gonadlar ilkel germinal hücreleri izole etmek için nasıl gösterir.

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-and-derivation-of-mouse-embryonic-germinal-cells

Research

JoVE Journal

Biology

16.1K Görüntüleme.  Mississippi State University. Ilkel germinal hücrelerin ayırt etmek için erken geliştirme aşamasında, embriyonik germinal hücreler, kanser ve kısırlık hakkındaki hipotezi adresine yeteneği mükemmel bir model. Bu protokol, 10,5-11,5 gün coitum fare embriyoları, gelişmekte olan gonadlar ilkel germinal hücreleri izole etmek için nasıl gösterir.

Video: Isolation and Derivation of Mouse Embryonic Germinal Cells

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 ...

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Yetişkin ve Embriyonik İskelet, Kas Microexplant Kültür ve İskelet Kası Kök Hücre İzolasyonu

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Isolation and Culture of Avian Embryonic Valvular Progenitor Cells

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of endocardium surrounded by myocardium. The myocardium secretes cardiac jelly, a hyaluronan-rich gelatinous matrix, into the atrioventricular (AV) junction and outflow tract (OFT) lumen. At stage HH14 valvulogenesis begins when a subset of endocardial cells receive signals from the myocardium, undergo endocardial to mesenchymal transformation (EMT), and invade the cardiac jelly. At stage HH25 the valvular cushions are fully mesenchymalized, and it is this mesenchyme that eventually forms the valvular and septal apparatus of the heart. Understanding the mechanisms that initiate and modulate the process of EMT and cell differentiation are important because of their connection to serious congenital heart defects. In this study we present methods to isolate pre-EMT endocardial and post-EMT mesenchymal cells, which are the two different cell phenotypes of the prevalvular cushion. Pre-EMT endocardial cells can be cultured with or without the myocardium. Post-EMT AV cushion mesenchymal cells can be cultured inside mechanically constrained or stress-free collagen gels. These 3D in vitro models mimic key valvular morphogenic events and are useful for deconstructing the mechanisms of early and late stage valvulogenesis.

This article will provide a method for isolating and culturing quail or chicken HH14- valve endocardial cells and HH25 valve cushion mesenchymal cells.

Kuş Embriyonik Kapak Progenitör Hücreler izolasyonu ve Kültür

Proper formation and function of embryonic heart valves is critical for developmental progression. The early embryonic heart is a U-shaped tube of ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embriyonik Fare Böbrek Nefrojenik Zone Hücreleri İzolasyon ve Kültür

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

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 ...

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it is not easy to establish an effective system for culturing these cells. Since the culture conditions can influence gene expression that confers pluripotency in hESCs and hiPSCs, the optimization and standardization of the culture method is crucial.
The establishment of hESC lines was first described by using MEFs as feeder cells and fetal bovine serum (FBS)-containing culture medium2. Next, FBS was replaced with knockout serum replacement (KSR) and FGF2, which enhances proliferation of hESCs3. Finally, feeder-free culture systems enable culturing cells on Matrigel-coated plates in KSR-containing conditioned medium (medium conditioned by MEFs)4. Subsequently, hESCs culture conditions have moved towards feeder-free culture in chemically defined conditions5-7. Moreover, to avoid the potential contamination by pathogens and animal proteins culture methods using xeno-free components have been established8.
To obtain improved conditions mouse feeder cells have been replaced with human cell lines (e.g. fetal muscle and skin cells9, adult skin cells10, foreskin fibroblasts11-12, amniotic mesenchymal cells13). However, the efficiency of maintaining undifferentiated hESCs using human foreskin fibroblast-derived feeder layers is not as high as that from mouse feeder cells due to the lower level of secretion of Activin A14. Obviously, there is an evident difference in growth factor production by mouse and human feeder cells.
Analyses of the transcriptomes of mouse and human feeder cells revealed significant differences between supportive and non-supportive cells. Exogenous FGF2 is crucial for maintaining self-renewal of hESCs and hiPSCs, and has been identified as a key factor regulating the expression of Tgf&beta;1, Activin A and Gremlin (a BMP antagonist) in feeder cells. Activin A has been shown to induce the expression of OCT4, SOX2, and NANOG in hESCs15-16.
For long-term culture, hESCs and hiPSCs can be grown on mitotically inactivated MEFs or under feeder-free conditions in MEF-CM (MEF-Conditioned Medium) on Matrigel-coated plates to maintain their undifferentiated state. Success of both culture conditions fully depends on the quality of the feeder cells, since they directly affect the growth of hESCs.
Here, we present an optimized method for the isolation and culture of mouse embryonic fibroblasts (MEFs), preparation of conditioned medium (CM) and enzyme-linked immunosorbent assay (ELISA) to assess the levels of Activin A within the media.

The quality of mouse embryonic fibroblasts (MEFs) is dictated by the right strain of mouse such as CF-1. Pluripotency-supportive MEFs and conditioned media (CM) obtained from these should contain optimal concentrations of Activin A, Gremlin and Tgf&beta;1 needed for the Activin/Nodal and FGF pathways to co-operatively maintain self-renewal and pluripotency.

Kültürleme İnsan Embriyonik ve Bağlı Pluripotent Kök Hücrelerinin için uygundur Fare embriyonik fibroblast hücreleri hazırlanması

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

Isolation of Embryonic Ventricular Endothelial Cells

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines offer significant advantages for their ease of use, these cell lines are unavailable for all potential cell types. By isolating primary cells from a specific region of interest, particularly from a transgenic mouse, more nuanced studies can be performed. The basic technique involves dissecting the organ or partial organ of interest (e.g. the heart or a specific region of the heart) and dissociating the organ to single cells. These cells are then incubated with magnetic beads conjugated to an antibody that recognizes the cell type of interest. The cells of interest can then be isolated with the use of a magnet, with a short trypsin incubation dissociating the cells from the beads. These isolated cells can then be cultured and analyzed as desired. This technique was originally designed for adult mouse organs but can be easily scaled down for use with embryonic organs, as demonstrated herein. Because our interest is in the developing coronary vasculature, we wanted to study this population of cells during specific embryonic stages. Thus, the original protocol had to be modified to be compatible with the small size of the embryonic ventricles and the low potential yield of endothelial cells at these developmental stages. Utilizing this scaled-down approach, we have assessed coronary plexus remodeling in transgenic embryonic ventricular endothelial cells.

Primary cell culture is a useful technique for analyzing specific populations of cells, particularly from transgenic mouse embryos at specific developmental stages. Herein, embryonic ventricles are dissected and dissociated, and antibody-conjugated beads recognize and separate out the endothelial cells for further analysis.

Embriyonik Ventriküler Endotel Hücre İzolasyonu

Cell culture has greatly enhanced our ability to assess individual populations of cells under myriad culture conditions. While immortalized cell lines ...

Isolation and Culture of Mouse Primary Pancreatic Acinar Cells

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one week. More than 20 x 106 acinar cells can be obtained from a single murine pancreas. This protocol offers the possibility to independently process as many as 10 pancreases in parallel. Because it preserves acinar architecture, this model is well suited for studying the physiology of the exocrine pancreas in vitro in contrast to cell lines established from pancreatic tumors, which display many genetic alterations resulting in partial or total loss of their acinar differentiation.

In this publication, we describe a rapid and convenient procedure for isolating and culturing primary pancreatic acinar cells from the murine pancreas. This method constitutes a valuable approach to study the physiology of fresh primary normal/untransformed exocrine pancreatic cells.

Fare Primer Pankreas Asiner Hücreleri İzolasyon ve Kültür

This protocol permits rapid isolation (in less than 1 hr) of murine pancreatic acini, making it possible to maintain them in culture for more than one ...