Bu çalışma kısmen, Ulusal Sağlık Enstitüsü (RO1 NS059043 ve RO1 ES015988), Ulusal Multipl Skleroz Derneği, Anemi Araştırma, Feldstein Tıp Vakfı Roche Vakfı ve Çocuk Shriners Hastaneler WD hibe tarafından desteklenmiştir. Biz Dr David Pleasure ve Jennifer Düzlem önerileriniz için teşekkür etmek istiyorum. Biz bu makalede ilgili hiçbir çatışan çıkarları beyan ederim.

GFP-Olig2 fare embriyonik kök hücreleri (mESCs) oligodendrosit öncü hücreler (OPC) üreten detaylı prosedürler: <ol><li> Fare ES hücre hattı GFP-Olig2 (G-Olig2), American Type Culture Collection (ATCC) satın alınmaktadır. MESCs ışınlanmış bir fare embriyonik fibroblast (MEF) besleyici tabaka üzerine 3 gün rutin olarak her geçişli. MEF hücreleri jelatin kaplı altı kuyu doku kültürü plaka EKH Pasajlanması önce en az bir gün% 0.1 kaplanmıştır. Mesc kültür orta,% 20 fetal sığır serumu (Gibco), 2 mM L-glutamin, 1 mM sodyum piruvat, 0,1 mM β-merkaptoetanol /,% 1 gerekli olmayan amino asitler (ile desteklenmiş Dulbecco&#39;nun modifiye Eagle Kullanıcı orta (DMEM) (Gibco) NEAA) ve 1000 U / ml lösemi inhibitör faktör. </li><li> MES koloniler tek hücre içine tripsinize (TrypLE, 5 dk, 37 ° C) ve nakavt serum replasmanı (KSR) orta askıya alınır, daha sonra hücrelerin bir hücre Costar ultra-düşük ek altı plaka (Corning) aktarılır 50.000 hücre / cm 2 yoğunluğu. Bu koşullarda, mESCs embriyoid organları (EBS) oluşturur. KSR orta,% 20 KSR, 1 mM sodyum piruvat,% 1 NEAA, 0.1 mM β-mercaptoethanol / ile desteklenmiş-minimal temel orta (MEM) oluşur. </li><li> Günden itibaren 7 gün 4, retinoik asit (RA, 0.2 M) ve purmorphamine (Pur, 1 M) Şekil dahildir. KSR veya N2 orta 1A. N2 orta 1X N2 takviyesi, 1 mM sodyum piruvat,% 1 NEAA ve 0.1 mM β-mercaptoethanol / içeren-MEM. Orta günlük değiştirildi. </li><li> 8 gün, EBS, N2, orta ve fibroblast oluşur OPC orta% 0.01 polyornithine kaplı yemekleri TrypLE (5 dk, 37 ° C) kaplanmıştır kullanarak ayrıştırılmıştır büyüme faktörü-2 (FGF-2, 20 ng / ml). Orta olmak üzere iki günde bir değiştirilir. </li><li> Hücreleri tripsinize (TrypLE, 3 dk, 37 ° C) ve konfluent olunca her hafta yaklaşık replated. 30 gün, hücrelerin% 80 GFP/Olig2 ifade, NG2 pozitif OPC, karakteristik. </li><li> Spike OPC oluşturmak için BacMam Na + kanallı kit bu Mesc türetilen OPC Na v 1.2 altbirim tanıtmak amacıyla kullanılmıştır. BacMam reaktif (1 ml, komponent A), 5 ml nihai hacmi PBS ile karıştırılır. Daha sonra, karma BacMam reaktif önce PBS ile çalkalayın (% 50-70 izdiham) 60 mm kültür çanak Mesc türetilmiş bir OPC kültürü içine eklenir. 2 saat inkübasyondan sonra, karma BacMam reaktif 1X arttırıcı ile desteklenmiş OPC orta ile kaldırılır ve değiştirilir. Arttırıcı bileşeni B DMSO (component C) sulandırılmış. Sonra, ilave 2 saat inkübasyon sonra tam OPC orta arttırıcı ile orta kaldırılır ve değiştirilir. Hücrelerin 24 saat sonra test. </li></ol> Temsilcisi Sonuçlar: Şekil farklılaşma protokolü takiben. 1A, G-Olig2 mESCs başlangıçta embriyoid organları (EBS) (Şekil 1B) oluşturmak için KSR orta ve 4 gün süreyle askıya alındı. Sonra, sırayla EBS RA ve Pur ile tedavi. EBS 4. Gün herhangi bir GFP floresan ve D8 (Şekil 1B) güçlü GFP floresan ifade vermedi. Bu noktada, EBS tripsinize ve büyüme faktörleri FGF-2 ile desteklenmiş N2 orta kaplanmış. 22 gün (D30) için daha fazla kültür sonra, GFP + hücre yüzdesi 80.3 ulaştı ± flow sitometri sonuçlarına göre% 0.6. Şekil. 1C, GFP + hücreler sürekli NG2 boyama (% 96.4 ± 1.3 GFP pozitif hücreler pozitif NG2 ve% 94.8 ± 1.5 NG2 pozitif hücreler, GFP pozitif idi) ile çakışıyordu. Böylece, bu hücreler OPC olarak tanımlayarak, GFP/Olig2 ve NG2 ifade. Mesc türetilen OPC (76 hücreleri) depolarizasyon (Şekil 2A) üzerine aksiyon potansiyelleri yangın başarısız oldu. Bakülovirüs Na v 1.2 altbirim taşıyan bir uyum sonra, bu Mesc türetilen OPC (17 hücrelerinin% 94.1, 16) spike (Şekil 2B) olarak sınıflandırılabilir. <img alt="Şekil 1" src="/files/ftp_upload/1960/1960fig1.jpg" /> Şekil 1. Embriyoid vücut protokol gösteren OPC içine GOlig-Mesc Farklılaşma (A) Programı (EB) tabanlı ve küçük molekül odaklı farklılaşma. D8, EBS ayrıştırılmış ve kaplanmıştır. Konfluent oldu hücreleri haftada bir pasajlandı. (B) D8, ama D4 EBS, GFP ifade gösterdi. (C) NG2 (kırmızı) immün D30 GFP (yeşil) ifadesi ile uyumlu idi. DAPI (mavi), çekirdekleri tanımlamak için kullanılır. Ölçek çubuğu: 20 mm. <img alt="Şekil 2" src="/files/ftp_upload/1960/1960fig2.jpg" /> Şekil 2. Mesc türetilen OPC nonspiking, OPC spike nesil virüs aracılı hücre 0 depolarize bile Na v kanal ifade (A) membran potansiyeli -60 mV ve Mesc türevi OPC yapıldı, aksiyon potansiyelleri yangın başarısız mV. (B) Mesc türetilen OPC bir örnekviral transdüksiyon sonra ateş aksiyon potansiyeli (kırmızı vurgulanmıştır).

<ol>
	<li>Xian, H., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dividing+Olig2-expressing+progenitor+cells+derived+from+ES+cells.">Dividing Olig2-expressing progenitor cells derived from ES cells.</a> Glia. 47 (1), 88-101 (2004).</li><li>Clair, A., Gottlieb, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+subset+of+ES-cell-derived+neural+cells+marked+by+gene+targeting.">A subset of ES-cell-derived neural cells marked by gene targeting.</a> Stem Cells. 21 (1), 41-49 (2003).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154-156 (1981).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1147 (1998).</li><li>Shin, S., Xue, H., Mattson, M. P., Rao, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stage-dependent+Olig2+expression+in+motor+neurons+and+oligodendrocytes+differentiated+from+embryonic+stem+cells.">Stage-dependent Olig2 expression in motor neurons and oligodendrocytes differentiated from embryonic stem cells.</a> Stem Cells Dev. 16, 131-141 (2007).</li><li>Billon, N., Jolicoeur, C., Ying, Q. L., Smith, A., Raff, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+timing+of+oligodendrocyte+development+from+genetically+engineered,+lineage-selectable+mouse+ES+cells.">Normal timing of oligodendrocyte development from genetically engineered, lineage-selectable mouse ES cells.</a> J Cell Sci. 115, 3657-3665 (2002).</li><li>Nishiyama, A., Lin, X. H., Giese, N., Heldin, C. H., Stallcup, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-localization+of+NG2+proteoglycan+and+PDGF+alpha-receptor+on+O2A+progenitor+cells+in+the+developing+rat+brain.">Co-localization of NG2 proteoglycan and PDGF alpha-receptor on O2A progenitor cells in the developing rat brain.</a> J Neurosci Res. 43, 299-314 (1996).</li><li>Ligon, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+NG2+neural+progenitor+cells+requires+Olig+gene+function.">Development of NG2 neural progenitor cells requires Olig gene function.</a> Proc Natl Acad Sci U S A. 103, 7853-7858 (2006).</li><li>Dawson, M. R., Polito, A., Levine, J. M., Reynolds, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-expressing+glial+progenitor+cells:+an+abundant+and+widespread+population+of+cycling+cells+in+the+adult+rat+CNS.">NG2-expressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS.</a> Mol Cell Neurosci. 24, 476-488 (2003).</li><li>Chittajallu, R., Aguirre, A., Gallo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NG2-positive+cells+in+the+mouse+white+and+grey+matter+display+distinct+physiological+properties.">NG2-positive cells in the mouse white and grey matter display distinct physiological properties.</a> J Physiol. 561, 109-122 (2004).</li><li>Karadottir, R., Hamilton, N. B., Bakiri, Y., Attwell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spiking+and+nonspiking+classes+of+oligodendrocyte+precursor+glia+in+CNS+white+matter.">Spiking and nonspiking classes of oligodendrocyte precursor glia in CNS white matter.</a> Nat Neurosci. 11, 450-456 (2008).</li></ol>

Oligodendrosit şartname de dahil olmak üzere, erken memeli geliştirme eğitimi için bir in vitro model sistemi sağlayarak, blastokist embriyo izole Embriyonik kök hücreler (EKH), 3, 4, organizmanın tüm hücre soyları ayırt edebilirsiniz. mESCs sonik kirpi (Shh) 5 tedavisinde oligodendrosit öncü hücreler (OPC) ayırt etmek için gösterilmiştir. Ayrıca, mESCs Shh kaynaklı OPC farklılaşma, embriyonik gelişim 6 gözlenen doğru zamanlama korur . Bu nedenle, in vitro OPC farklılaşmasında mESCs doğası in vivo geliştirme öğrendim ne ile tutarlı olarak kabul edilir. Burada, küçük moleküller RA ve Shh agonist Pur kullanarak, yüksek verimlilik ile GFP + Olig2 + NG2 + OPC başarıyla G-Olig2 mESCs farklılaşmış. Toplam hücrelerinin önemli bir yüzdesini oluşturan (~% 5) ve proteoglikan NG2 7 ifadesi ve helix-loop-helix transkripsiyon faktörü Olig2 8 ile karakterize OPC, gelişmekte olan ve olgun MSS oligodendrosit oluşturmak ana prolifere hücre tipi 9. V kanal Na göstermiştir yaklaşık iki yıl araştırmacılar için bir OPC ICSI&#39;nin ifade depolarizasyon 10, 11 üzerine devreye sokulabilir. Ayrıca, son çarpıcı bir gözlem 9 beyaz cevher in situ sıçan MSS, voltaj kapılı sodyum (Na V) kanalları, ifade bağlı olarak zaman depolarize, OPC (~% 50) aksiyon potansiyelleri oluşturulur ve böylece spike bölünmüştür olabilir alt popülasyonlar nonspiking. Ancak, bu spiking özellikleri fonksiyonları halen büyük ölçüde bilinmiyor. Biz elektrofizyolojik, Shh bağımlı protokol ile farklılaşmış Mesc kaynaklı OPC, in situ beyin OPC olarak aynı değildi, o bulundu . Subunit Na V 1.2 tanıttıktan sonra, bu sessiz Mesc türetilen OPC spike kabil edildi. Böylece, spike / Mesc türetilen OPC ve farklılaşma protokol burada açıklanan nonspiking (1) spike ve nonspiking OPC arasındaki işlevsel farklılıklar, (2) Mesc kaynaklı OPC, sodyum kanal ifade teşvik tarama yeni faktörler eğitimi kolaylaştırmak ( 3) geliştirilmesi ve insan ESC veya uyarılan pluripotent kök hücreleri (iPSCs) OPC farklılaşma protokolü optimize.

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differentiation of embryonic stem cells into oligodendrocyte precursors

Oligodendrosit, merkezi sinir sistemi myelinating hücreleri vardır. Demiyelinizan hastalıkları rejeneratif hücre tedavisi için, saf bir nüfus nakli için soy taahhüt oligodendrosit öncü hücreler (OPC) kaynaklanan önemli bir ilgi var. OPC, transkripsiyon faktörü Olig2 ve proteoglikan NG2 yüzey ifade aktivitesi ile karakterize edilir. GFP-Olig2 (G-Olig2) fare embriyonik kök hücre (Mesc) muhabiri hattı kullanarak, GFP + Olig2 + NG2 + OPC içine mESCs farklılaşması için koşulları optimize edilmiş. Bizim protokolde, ilk mESCs embriyoid organlarının nesil (EBS) açıklar. İkincisi, küçük moleküller Mesc türetilen EBS tedavi açıklar: (1) retinoik asit (RA) ve (2) belirlenen kültür koşulları altında sonik kirpi (Shh) agonist purmorphamine (Pur) oligodendroglial soy içine doğrudan EB farklılaşma. Bu yaklaşım, OPC 30 gün bir süre yüksek verim (&gt;% 80) ile elde edilebilir. Bu protokolde mESCs türetilen hücreler primer doku kültürü ile elde edilen OPC fenotipik benzer. Mesc türetilen OPC in situ beyin OPC bir ICSI&#39;nin açıklanan spike özelliği görünmüyor. Bu elektrofizyolojik özellik çalışma için, ektopik Na ifade ederek Mesc-türetilen OPC spike nesil tarif<sub&gt; V</sub&gt; 1.2 altbirimlerine. Bu protokol elde spike ve nonspiking hücreleri OPC iki alt popülasyonlar üzerinde önceden fonksiyonel çalışmalara yardımcı olacaktır.

Watch this Scientific Journal Video about Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors at JoVE.com

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Biz küçük bir molekül tabanlı oligodendrosit öncü hücreler (OPC) içine fare embriyonik kök hücreleri farklılaşma için protokol açıklar. Bu protokol, 30 gün farklılaşma, yüksek verimlilik ile Olig2 + NG2 + OPC üretir. Ayrıca, aksiyon potansiyelleri ateş &quot;spike&quot; OPC oluşturmak için bir yöntem açıklanmaktadır.

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 ...

differentiation-embryonic-stem-cells-into-oligodendrocyte

Research

JoVE Journal

Biology

15.2K Görüntüleme.  School of Medicine, University of California, Davis. Biz küçük bir molekül tabanlı oligodendrosit öncü hücreler (OPC) içine fare embriyonik kök hücreleri farklılaşma için protokol açıklar. Bu protokol, 30 gün farklılaşma, yüksek verimlilik ile Olig2 + NG2 + OPC üretir. Ayrıca, aksiyon potansiyelleri ateş "spike" OPC oluşturmak için bir yöntem açıklanmaktadır.

Video: Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

İnsan Embriyonik Kök Hücre (ES) yayılması

Biyoloji

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Fare Nöral Kök Hücre Öncüleri Kültür

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

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 ...

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 ...

In vitro Differentiation of Mouse Embryonic Stem (mES) Cells Using the Hanging Drop Method

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either remain a stem cell or become another type of cell with a more specialized function, This promising of science is leading scientists to investigate the possibility of cell-based therapies to treat disease.

When culture in suspension without antidifferentiation factors, embryonic stem cells spontaneously differentiate and form three-dimensional multicellular aggregates. These cell aggregates are called embryoid bodies(EB). Hanging drop culture is a widely used EB formation induction method. The rounded bottom of hanging drop allows the aggregation of ES cells which can provide mES cells a good environment for forming EBs. The number of ES cells aggregatied in a hanging drop can be controlled by varying the number of cells in the initial cell suspension to be hung as a drop from the lid of Petri dish. Using this method we can reproducibly form homogeneous EBs from a predetermined number of ES cells. 

In vitro Differentiation of Mouse Embryonic Stem mES Cells Using the Hanging Drop Method

This video demonstrates how to conduct in vitro differentiation of mouse embryonic stem cells to embryoid bodies using the hanging drop method.

Asma Bırak Yöntemiyle Fare Embriyonik Kök (MES) Hücreler in vitro Farklılaşma

Stem cells have the remarkable potential to develop into many different cell types. When a stem cell divides, each new cell has the potential to either ...

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 ...

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 ...

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 ...

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

Studying germ cell formation and differentiation has traditionally been very difficult due to low cell numbers and their location deep within developing embryos. The availability of a "closed" in vitro based system could prove invaluable for our understanding of gametogenesis. The formation of oocyte-like cells (OLCs) from somatic stem cells, isolated from newborn mouse skin, has been demonstrated and can be visualized in this video protocol. The resulting OLCs express various markers consistent with oocytes such as Oct4 , Vasa , Bmp15, and Scp3. However, they remain unable to undergo maturation or fertilization due to a failure to complete meiosis. This protocol will provide a system that is useful for studying the early stage formation and differentiation of germ cells into more mature gametes. During early differentiation the number of cells expressing Oct4 (potential germ-like cells) reaches ~5%, however currently the formation of OLCs remains relatively inefficient. The protocol is relatively straight forward though special care should be taken to ensure the starting cell population is healthy and at an early passage.

Differentiation of Newborn Mouse Skin Derived Stem Cells into Germ-like Cells In vitro

In recent years the formation of germ cells using in vitro culture methods has been demonstrated using several different types of somatic stem cells. This article will visually present the differentiation of newborn mouse derived stem cells to early oocyte-like cells.

Germ benzeri hücreler içine Yenidoğan Fare Cilt Türetilmiş Kök Hücre Farklılaşma<em&gt; In vitro</em

Studying  germ cell formation and differentiation has traditionally been very difficult  due to low cell numbers and their location deep within developing ...