Yazarlar Andrea Axtell, Satoshi Itoh, MD, Jeff Velotta, MD, Hibe Hoyt, Robert C. Robbins, MD, Jin Yu, MD, Tim Doyle, Doktora ve teknik yardım için Stanford Küçük Hayvan Imaging Core teşekkür ederim. Yazarlar ayrıca veteriner ekipman desteği için teşekkür ederiz. AM Bickford, Inc. Bu araştırma, Ulusal Sağlık Enstitüleri araştırma hibeler (R01 HL-75.774, R01 CA098303, R21 HL085743 1K12 HL087746), California University of California (15IT-0257 ve 1514RT-0169) Tütün İlgili Hastalık Araştırma Programı tarafından desteklenen ve Rejeneratif Tıp California Institute (RS1-00.183). NH bir burs Amerikan Kalp Derneği tarafından desteklenmektedir. Kalp Derneği.

1. Endotel hücreleri içine fare EKH farklılaşma <ol><li> Endotel hücreleri EKH ayırt için protokol başka açıklanan ve bu protokol 2,3 odak değildir. Kısaca, hücrelerin ayırt etmek için izin verilir ve floresan aktif hücre sıralama (FACS), CD31 ya da vasküler endotelyal kaderin (VE-kaderin) gibi endotel işaretleri için pozitif hücreler daha sonra arıtılmış. </li></ol> 2. Double Fusion Reporter Gen ve lentiviral İletimi İnşaatı <ol><li> Biyoparlaklık ateşböceği lusiferaz (fluc) muhabiri genlerin ifade etmek için değiştirilmiş hücreler izlemek için kullanılabilir. Bu uygulama için, hücreleri bir iç ubikuitin organizatörü kontrol altında fluc ve gelişmiş hem de yeşil floresan protein (eGFP) füzyon gen içerir. Hücreleri değiştirmek için optimize önemli genetik unsurları içeren transgen lentiviral vektör yerine getirmek için biyo-güvenlik kriterleri artan transdüksiyon etkinliğinin yanı sıra, laboratuarda fluc eGFP füzyon muhabiri geni taşıyan geliştirilen PFU-FG vektör stabil bir şekilde kullanılabilir sonra hücrelerin farklılaşması transduce. Bu füzyon inşa nakledilen hücrelerin biyoparlaklık ve floresan izleme sağlar. Virüs parçacıkları, iletimi ve ifade muhabiri genlerin başka bir yerde 4 açıklanmıştır etiketli hücrelerinin üretimi üretmek için prosedür. </li></ol> 3. Iskemik hindlimb Transplantasyon ESC-endotel hücreleri <ol><li> Hücre nakli için hindlimb iskemi geçirdiği bir fare hazırlama prosedürü başlayın. Bunu yapmak için, anestezi indüksiyon odasına 1L/min bir akış hızında% 100 oksijen izofluran% 1-3 içeren fare yerleştirin. </li><li> Dış uyaranlara tepkisiz kadar indüksiyon odasında fare bırakın. Ardından indüksiyon odasından hayvan çıkarın. </li><li> Daha sonra, işletim masaya yatar pozisyonda hayvan yer ve 1L/min bir akış hızında% 100 oksijen içinde% 1-3 izofluran sürekli bir akış bağlamak. </li><li> Üç alternatif betadin ve alkol scrubs hindlimb cilt silin. </li><li> Cilt temizlendikten sonra fosfat 30 mcL bir milyon ESC edilen endotel hücreleri elde tamponlu salin (PBS). 28 iğne bu hücrelerin yükleyin. </li><li> Hücrelerin hazır olduğunuzda, hafifçe kaldırın ve gastroknemius kas yerini daha iyi görselleştirmek için hindlimb uzatmak. Bacak uzatılmış olmakla birlikte, altta yatan kas içine deri yoluyla iğne takın. Kemik yaklaşım değil dikkat edin. Ve yavaşça gastroknemius 30 mcL hücre karışımı enjekte edilir. Intramüsküler enjeksiyon için, 30 mcL hacim enjekte edilebilir sınırı yakın. Deneyimlerimiz, şırınga iğneleri kaybı hacmi yok, çünkü nedenle, 28-gauge insülin şırıngaları tercih edilmektedir. </li><li> Enjeksiyon işlemi tamamlandıktan sonra, kurtarma kafesine fare dönmek ve uyanık kadar sürekli izlemek. Hayvan kurtarmak ve birkaç saat sonra nakledilen hücrelerin in vivo biyoparlaklık görüntüleme ile devam etmek için izin verin. </li></ol> 4. ESC edilen endotel hücreleri in vivo görüntüleme Biyoparlaklık <ol><li> Nakledilen ESC-türetilmiş endothelials hücreleri, bir iç ubikuitin organizatörü kontrol altında fluc ve eGFP füzyon gen ifade etmek için modifiye edilmiştir. Bu nedenle, biyoparlaklık iskemik hindlimb içinde hücreler izlemek için kullanılabilir. </li><li> Bu adımı başlamak için, biyoparlaklık görüntüleme sistemi ve Yaşam Görüntü toplama yazılımı açın. Sonra toplama sistemi başlatmak ve görüş alanının boyutlarını belirtmek. </li><li> Arka plan emisyon emmek için görüntüleme kutusu üzerine, yer siyah, mat kağıt. </li><li> Görüntüleme kutusu hazır olduğunda fare 1L/min çıkış izofluran% 1-3 olarak oksijen içeren anestezi indüksiyon odasına koyun. Dış uyaranlara tepkisiz kadar indüksiyon odasında fare bırakın. Ardından indüksiyon odasından hayvan çıkarın. </li><li> Gerektiği gibi bir elektrikli tıraş makinesi ile her iki arka ayaklarında saç çıkarın. </li><li> D-luciferase vücut ağırlığının gram başına 10 mcL periton içine enjekte edilir. D-luciferase 15 mg / mL PBS filtre stok çözelti içine önceden hazırlanmış. </li><li> Luciferase enjekte edildikten sonra, hayvan izofluran, sürekli bir akış bağlı yatar pozisyonda siyah kağıt üzerinde görüntüleme kutuya koyun. </li><li> Hayvan görüntü kutunun içine yerleştirilmesi ve izofluran için bağlanma. </li><li> Görüntü doymuş olduğu bir optimum pozlama süresini belirlemek için 10-60 saniye görüntüleri elde etmek için Başlat. Görüntü doymuş hale gelirse, pozlama süresini azaltmak. Biyoparlaklık sinyal çok zayıfsa, pozlama süresi artar. </li><li> Istimalsinyal maksimum ve sonra kaybolur ulaşıncaya kadar g en uygun pozlama süresi, resimlerin her 1-3 dakikada edinmeye devam. Satın alma işlemi tamamlandığında, dosyayı kaydedin. </li><li> Verileri analiz etmek, enjeksiyon yerinde kapak ilgi alanları (ROI) seçin. Negatif kontrol olarak, benzer bir yatırım getirisi olmayan çalışan bacak için seçilebilir. Yazılımını kullanarak, her timepoint ROI&#39;ler fotonlar / saniye / cm 2 / steradian (p / s / cm 2 / sr) birimleri cinsinden ifade edilir toplam parlaklığı, ölçer. Maksimum değeri son verileri kullanılmalıdır. Veriler de ileride kullanmak üzere, Excel elektronik tablo ihraç edilebilir. </li><li> Bir kez tüm veriler elde edilir, hayvan kurtarma kafes ve hayvan uyanır kadar sürekli izlemek. </li><li> Hücreler zaman içinde izlemek için bu prosedürü tekrarlayın. </li><li> Istenilen bir zaman noktasında, hayvan doku fonksiyon değerlendirilmesi için ötenazi olabilir. </li></ol> 5. Temsilcisi Sonuçlar <img src="/files/ftp_upload/old/1034_2009_1_22_4/figure 1.png" alt="Şekil 1" width="600" height="600" /> Sol iskemik hindlimb nakledilen hücrelerin bir temsilcisi biyoparlaklık görüntü Şekil 1&#39;de gösterilmiştir. Biyoparlaklık satın alınması sırasında, şiddeti zamanla artacak ve zaman seyri sırasında elde edilen maksimum değeri son değer olarak rapor edilmelidir.

<ol>
	<li>Niiyama, H., Huang, N. F., Rollins, M., Cooke, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Murine+model+of+hindlimb+ischemia.">Murine model of hindlimb ischemia.</a> JoVE. , (2008).</li><li>Levenberg, S., Golub, J. S., Amit, M., Itsakovitz-Eldor, J., Langer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+derived+from+human+embryonic+stem+cells.">Endothelial cells derived from human embryonic stem cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 4391-4396 (2002).</li><li>Yamashita, J., Itoh, H., Hirashima, M., Ogawa, M., Nishikawa, S., Yurugi, T., Naito, M., Nakao, K., Nishikawa, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flk1-positive+cells+derived+from+embryonic+stem+cells+serve+as+vascular+progenitors.">Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors.</a> Nature. 408, 926-926 (2000).</li><li>De, A., Yaghoubi, S. S., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+lentiviral+vectors+in+noninvasive+molecular+imaging.">Applications of lentiviral vectors in noninvasive molecular imaging.</a> Methods Mol Biol. 433, 177-202 (2008).</li><li>Niiyama, H., Kai, H., Yamamoto, T., Shimada, T., Sasaki, K., Murohara, T., Egashira, K., Imaizumi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+endogenous+monocyte+chemoattractant+protein-1+in+ischemia-induced+neovascularization.">Roles of endogenous monocyte chemoattractant protein-1 in ischemia-induced neovascularization.</a> J. Am. Coll. Cardiol. 44, 661-666 (2004).</li><li>Cook, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+anatomy+of+the+laboratory+mouse.">The anatomy of the laboratory mouse.</a> , (1976).</li><li>Contag, P. R., Olomu, I. N., Stevenson, D. K., Contag, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+indicators+in+living+mammals.">Bioluminescent indicators in living mammals.</a> Nature Med. 4, 245-247 (1998).</li><li>Ray, P., De, A., Min, J. J., Tsien, R. Y., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+tri-fusion+multimodality+reporter+gene+expression+in+living+subjects.">Imaging tri-fusion multimodality reporter gene expression in living subjects.</a> Cancer Res. 64, 1323-1330 (2004).</li><li>Huang, N. F., Lee, R. J., Li, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+physical+regulation+of+stem+cells+and+progenitor+cells:+potential+for+cardiovascular+tissue+engineering.">Chemical and physical regulation of stem cells and progenitor cells: potential for cardiovascular tissue engineering.</a> Tissue Eng. 13, 1809-1823 (2007).</li><li>Cao, F., Lin, S., Xie, X., Ray, P., Patel, M., Zhang, X., Drukker, M., Dylla, S. J., Connolly, A. J., Chen, X., Weissman, I. L., Gambhir, S. S., Wu, 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=In+vivo+visualization+of+embryonic+stem+cell+survival,+proliferation,+and+migration+after+cardiac+delivery.">In vivo visualization of embryonic stem cell survival, proliferation, and migration after cardiac delivery.</a> Circulation. 113, 1005-1114 (2006).</li><li>Wilson, K., Yu, J., Lee, A., Wu, 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=In+vitro+and+in+vivo+bioluminescence+reporter+gene+imaging+of+human+embryonic+stem+cells.">In vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells.</a> J Vis Exp. , (2008).</li></ol>

EKH farklılaşma ve endotel hücreleri de dahil olmak üzere her üç germ tabakasından oluşan hücre soylarının ortaya çıkmasına neden yeteneklerini plastisite nedeniyle doku iskemi tedavisi için umut vaat eden bir hücre kaynağı. EKH ile ilgili etik kaygıları aşmak için, bağlı pluripotent kök hücreleri (iPSCs) etik kaygılar üstesinden alternatif pluripotent kök hücre kaynağı olabilir. EKH yanı sıra, yetişkin kök hücreleri, endotel progenitör hücrelerin (EPC) ve hematopoetik kök hücreleri (HKH&#39;lerin) g...

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embryonic stem cell-derived endothelial cells for treatment of hindlimb ischemia

Bacaklar ve ayaklar, oksijenli kan ve besin kaynağı periferik arterlerin daralması periferik arter hastalığı (PAH), Bu patoloji aralıklı topallama (yürüme ağrısı), ağrılı iskemik ülserasyonlar, hatta ekstremite tehdit eden bir kangren gibi semptomlara neden olur. Genellikle, tüm kan ve lenf damarlarının luminal yüzeyi yatırım endotel hücreleri bir tek tabaka, damar endotel, vasküler homeostazı ve vasküler rejenerasyon baskın bir rol oynadığı düşünülmektedir. Sonuç olarak, endotel hücre tabanlı rejenerasyon kök PAD tedavi için umut verici bir yaklaşım olabilir. Bu video, embriyonik kök hücre nakli (ESC) göstermektedir elde edilen biyoparlaklık görüntüleme homing hücre ve hayatta kalma non-invaziv izleme takip PAD bir model olarak endotel hücreleri, tek taraflı hindimb iskemi tedavisi için. Hücre teslimat ve görüntüleme için özel malzemeler ve usulleri açıklanan olacaktır. Bu protokol Niiyama ark hindlimb iskemi indüksiyon açıklayan başka bir yayın izler. 1

Watch this Scientific Journal Video about Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia at JoVE.com

Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Teslimi için cerrahi işlem embriyonik kök hücre elde edilen endotel hücreleri iskemik hindlimb biyoparlaklık görüntüleme ile non-invaziv takibi ile gösterilmiştir.

Embriyonik Kök Hücre Tedavi Hindlimb İskemi Türetilmiş Endotel Hücreleri

Peripheral arterial disease (PAD) results from narrowing of the peripheral arteries that supply oxygenated blood and nutrients to the legs and feet, This ...

embryonic-stem-cell-derived-endothelial-cells-for-treatment-hindlimb

Research

JoVE Journal

Biology

11.7K Görüntüleme.  Stanford University. Teslimi için cerrahi işlem embriyonik kök hücre elde edilen endotel hücreleri iskemik hindlimb biyoparlaklık görüntüleme ile non-invaziv takibi ile gösterilmiştir.

Video: Embryonic Stem Cell-Derived Endothelial Cells for Treatment of Hindlimb Ischemia

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

İnsan Embriyonik Kök Hücre (ES) yayılması

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 ...

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

İnsan Embriyonik Kök Hücreleri Mitokondrial Kompleksi Aktivite araştırmayı

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

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 ...

Murine Model of Hindlimb Ischemia 

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide. Medical therapies for symptomatic relief are limited. Surgical or endovascular interventions are useful for some individuals, but long-term results are often disappointing. As a result, there is a need for developing new therapies to treat PAD. The murine hindlimb ischemia preparation is a model of PAD, and is useful for testing new therapies. When compared to other models of tissue ischemia such as coronary or cerebral artery ligation, femoral artery ligation provides for a simpler model of ischemic tissue. Other advantages of this model are the ease of access to the femoral artery and low mortality rate. 
In this video, we demonstrate the methodology for the murine model of unilateral hindimb ischemia. The specific materials and procedures for creating and evaluating the model will be described, including the assessment of limb perfusion by laser Doppler imaging. This protocol can also be utilized for the transplantation and non-invasive tracking of cells, which is demonstrated by Huang et al.1.

Murine Model of Hindlimb Ischemia

The surgical procedure for induction of unilateral hindlimb ischemia is demonstrated, with confirmation of ischemia by laser Doppler perfusion imaging.

Murin Model Hindlimb İskemi

In the United States, peripheral arterial disease (PAD) affects about 10 million individuals, and is also prevalent worldwide.  Medical therapies for ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Karyotipik İnsan Embriyonik Kök Hücreleri kromozom Yayılı Hazırlık

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

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

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

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

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

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

Embriyonik Kök Hücreler farklılaşma oligodendrosit Öncüleri

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Efficient Differentiation of Mouse Embryonic Stem Cells into Motor Neurons

Direct differentiation of embryonic stem (ES) cells into functional motor neurons represents a promising resource to study disease mechanisms, to screen new drug compounds, and to develop new therapies for motor neuron diseases such as spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Many current protocols use a combination of retinoic acid (RA) and sonic hedgehog (Shh) to differentiate mouse embryonic stem (mES) cells into motor neurons1-4. However, the differentiation efficiency of mES cells into motor neurons has only met with moderate success. We have developed a two-step differentiation protocol5 that significantly improves the differentiation efficiency compared with currently established protocols. The first step is to enhance the neuralization process by adding Noggin and fibroblast growth factors (FGFs). Noggin is a bone morphogenetic protein (BMP) antagonist and is implicated in neural induction according to the default model of neurogenesis and results in the formation of anterior neural patterning6. FGF signaling acts synergistically with Noggin in inducing neural tissue formation by promoting a posterior neural identity7-9. In this step, mES cells were primed with Noggin, bFGF, and FGF-8 for two days to promote differentiation towards neural lineages. The second step is to induce motor neuron specification. Noggin/FGFs exposed mES cells were incubated with RA and a Shh agonist, Smoothened agonist (SAG), for another 5 days to facilitate motor neuron generation. To monitor the differentiation of mESs into motor neurons, we used an ES cell line derived from a transgenic mouse expressing eGFP under the control of the motor neuron specific promoter Hb91. Using this robust protocol, we achieved 51&plusmn;0.8% of differentiation efficiency (n = 3; p &lt; 0.01, Student's t-test)5. Results from immunofluorescent staining showed that GFP+ cells express the motor neuron specific markers, Islet-1 and choline acetyltransferase (ChAT). Our two-step differentiation protocol provides an efficient way to differentiate mES cells into spinal motor neurons.

We developed a new protocol to improve efficiency of in vitro differentiation of mouse embryonic stem cells into motor neurons. The differentiated ES cells acquired motor neurons features as evidenced by expression of neuronal and motor neuron markers using immunohistochemical techniques.

Motor Nöronlar içine Fare Embriyonik Kök Hücre Verimli Farklılaşma

Direct differentiation of embryonic stem (ES) cells into functional  motor neurons represents a promising resource to study disease mechanisms, to  screen ...