Embriyonik Kök Hücre Farklılaşması <ol><li> ESC tripsin ile tedavi ile embriyonik kök hücreler (ESC) yakın bir konfluent balon toplayın. </li><li> Farklılaşma medya 5 ml hücreleri yeniden süspanse edin ve olmayan bir-jelatin kaplı T25 transfer. 37 ° C&#39;de 45 dakika için. Süpernatant Al ve santrifüj yoluyla hücreleri toplamak. Not: ESC kültürlerden gelen Fare embriyonik fibroblastlar (MEFS) olmayan jelatin kaplı T25 kolayca tutunur ve böylece bu kaplama adımı sırasında kültür tükenmiş. MEFS üzerinde kültür sizin ESC değilseniz, ön-plaka atlayabilirsiniz. </li><li> 333.333 cells/50 ml Farklılaşma medyada yeniden süspanse MEF-tükenmiş ESC. Bu yoğunlaşma, 100 ESC/15 ml sonuçlanır. </li><li> Kullanımı çok kanallı pipet, cells/15 15 cm 2 petri tabak 100 mL damla plaka ESC. 8 kanallı pipet ile, plaka başına 18-22 satır (plaka başına 5 ml hakkında) damla hakkında sığdırmak mümkün olmalıdır. Not: Bu protokol, damla 2-5 15 cm 2 yemekleri yeterli olandan daha fazla olacak ve 2-5 x 10 6 gün altı EB-kaynaklı hücrelerin boyun eğmek zorundadır . </li><li> Damla yavaşça ters plakaları çevirin. Için 37 ° C&#39;de% 5 CO 2 ile 48 saat inkübe edin . </li><li> Havuz yavaşça 15 ml konik tüp plakaları ve transfer dönen düşer. 4-6 mL PBS plakalar durulayın ve 15 ml konik aynı eklemek. Not: beş damla plakalar asılı havuzu olabilir. EBS farklılaştırmaya devam ederler ve onlar da yoğunlaştığı takdirde büyük öbekler şeklinde eğilimi artacaktır. </li><li> Ağırlık (yaklaşık 10 dakika) tarafından toplanmış EBS yerleşmek için izin verin. EBS rahatsız olmadan süpernatant aspire. 10 ml Farklılaşma medya ve 10 cm yapışmaz petri tabak transfer yavaşça tekrar süspansiyon haline getirin. </li><li> 50 rpm ve 37 ° inkübe plaka çalkalayıcı yerleştirin petri tabak ° C% 5 CO 2. </li><li> 24 saat sonra (farklılaşma dört gün), girdap plakaları petri merkezinde EBS konsantre. Dikkatle döviz, 5 ml taze Farklılaşma Medya ile medya% 50 (5 ml). İki gün daha inkübatör plakasını dönün. </li></ol> MSCV-HoxB4-IRES-GFP ile EB disosiasyon ve enfeksiyon <ol><li> Farklılaşma altı günü günde 15 ml konik tüp EBS aktarın. Yerçekimi ile yerleşmek için izin verin. </li><li> 10 ml PBS içinde aspire medya ve tekrar süspansiyon haline getirin. EBS yerçekimi ile yerleşmek için izin ver. Aspire PBS. </li><li> 250 ml disosiasyon enzim karışımı ve 1 mL PBS ekleyin. Bazen enzimler ve EBS karıştırmak için tüp dönen, 37 ° C su banyosunda 20 dakika inkübe edin. </li><li> 8 mL enzim disosiasyon tampon ekleyin. </li><li> EBS tamamen ayrışmış kadar karışımı (aşırı pipetleme EB-kaynaklı hücre hazırlama içinde ölüm artacak yaklaşık 10 kez) 5 ml pipet kullanarak Karışım. <ol><li> Karışım EBS ayrıştırmaları olarak hücreleri ile bulutlu olacak. </li><li> Öğütme sırasında konik tüp alt karşı pipet yerleştirmek disosiasyon büyük ölçüde kolaylaştıracak bir kuvvet oluşturacaktır. </li></ol></li><li> Santrifüj yoluyla hücreler toplayın. </li><li> 5 ml% 10 IMDM içinde süspanse edin hücreleri EB-türetilmiş ve tripan mavisi dışlama kullanarak saymak Not: farklılaşma gün dört ve altı gün arasında, EBS cavitate, altı günde, apoptoz ve hücre death.Thus büyük miktarda sonuçları EB-türetilen hücrelerin% 30 yukarı tripan mavi ile leke olabilir. </li><li> Süspanse edin EB-elde edilen 100.000 hücre hücre / 2 mL% 10 IMDM + viral supernatant gibi bir İçişleri Bakanlığı 5-10 nihai konsantrasyonu 8 mg / ml protamin sülfat achieved.Add. Not: plaka dört adet 6-iyi levhalar (2 ml / varsayarak) yeterli EB / viral supernatant karışımı hazırlayın . </li><li> Levha 2 mL EB / viral supernatant karışım başına iyi OP9 stroma hücreleri (aşağıya bakınız) ile ön kaplama dört 6 kuyucuğu. </li><li> 2500 devirde 90 dakika süreyle oda sıcaklığında plakaları santrifüjleyin. 37 ° inkübatör plakaları transfer ° C,% 5 CO 2. </li><li> 4-6 saat sonra, plakalardan supernatant hasat ve santrifüj yoluyla durdurulması kalan herhangi bir potansiyel hücreleri toplamak. </li><li> 48 ml% 10 IMDM yeniden süspanse pellet (küçük olabilir) + sitokinler. Uygulama 2 mL / enfeksiyon yapıldı ve süpernatant toplanan edildiği orijinal dört 6-kuyucuğu için tabanda. Not: Sürekli kullanım sırasında taze% 10 IMDM + sitokinler hazırlar. </li><li> Plakalar için 37 ° C&#39;de% 5 CO 2 ile yedi gün inkübe edin . Kolonileri gün dört sonrası enfeksiyon ve büyük ve yedi gün iyi biçimli görünür olmalıdır. Sağlam bir genişleme / yedi günde 40-60 koloniler boyun eğmek zorundadır. </li><li> Gün yedi post-enfeksiyon, tripsin ile tedavi bütün kuyulardan (OP9 stroma dahil) tüm hücreleri toplamak ve havuz. Not: süpernatant veya tripsin treatment.At Bu noktada kültür sırasında istihdam PBS yıkar atmayın, bazı koloniler sadece gevşek yapışmış olabilir ve birçok hücre floa vardırting suspension.Collect ve havuz tüm yıkar ve supernatant potansiyel olarak değerli hücreler atılır sağlamak. </li><li> 8 ml taze% 10 IMDM yeniden süspanse hücreler + sitokinler. 2 mL / şişesi dört T75 şişeler içine dağıtın. Her balona ilave bir 13 mL% 10 IMDM + sitokinler ekleyin. 37 ° ° C&#39;de üç gün için% 5 CO 2 (toplam on gün sonrası enfeksiyon). </li></ol> Kültür ve kaplama OP9 stroma <ol><li> % 20 a-MEM standart protokollere göre OP9 stroma koruyun. Confluency için yetiştirilen OP9 hücrelerin özellikleri değişecektir. Her zaman 1:3 &#39;ten daha fazla bölünmüş confluency% 80 ayrıldı. </li><li> 25.000 /% 20 a-MEM dört adet 6-iyi plakaları MSCV-HoxB4 IRES-GFP, plaka OP9 hücreleri ile altı gün EB-kaynaklı hücrelerin enfeksiyonu önce 24 saat. </li></ol> Koleksiyon, bölme ve transplantasyon <ol><li> Tripsin ile on gün ile tedavi için OP9 stroma hücreleri genişletilmiş toplayın. Hücreleri saymak. Not: süpernatant veya PBS atmayın tripsin tedavisi sırasında istihdam yıkar. Kültür, bu noktada, bazı koloniler sadece gevşek yapışmış olabilir ve suspension.Collect ve havuz, potansiyel olarak değerli hücreler atılır olduğundan emin olmak için tüm yıkar ve supernatant yüzen birçok hücre vardır. Not: iyi bir genişleme toplam 160-200 x 10 6 hücre, 40-50 x 10 6 hücre / enfekte orijinal 6-plaka arasında boyun eğmek zorundadır. </li><li> Hücreler protokolde bu noktada standart tekniklere göre manyetik boncuk seçimi veya FACS ile fraksiyone olabilir. </li><li> Transplantasyonu için retro-orbital veya kuyruk damar enjeksiyonu ile hücreler, ışınlama 2.5 saat 9.25 ji bölünmüş bir doza maruz Rag-2/gc eksik alıcıları (15-22 arasında gram ağırlığında) sunuyor. dışında. Not: hayvanların kilo ışınlama sırasında 15-22 gram arasında kalan KRİTİK. 22 gramdan büyük ise, uygun ablasyon 9.25 ji sağlamak ve hücreleri öldürücü radyasyon ve / veya aşılamak hematopoetik bölmesi kurtarma aracı olmayabilir yeterli doz ışınlama olmaz. Büyük hayvanların nakli, uygun ışınlama dozu deneysel olarak tespit edilmelidir. </li><li> 2-5 x 10 6 hücre / hayvan en az bir doz öldürücü ışınlama kurtarma garanti etmek için gereklidir . Hücreleri fraksiyonasyon ise, 2-5 x 10 6 hücre eşdeğerleri (örneğin hücre fraksiyone olmayan havuz ilgi nüfusunun% 10 temsil ediyorsa, 2-5 x 10 5 hücre / hayvan enjeksiyon) enjekte edilir. Not:&gt; 2 x 10 6 hücre enjekte DAİMA yüksek sayıda hücre retro-orbitally teslim edildiğinde neden olabilir yörüngede, teratom oluşumunu önlemek için kuyruk damar yoluyla enjekte. </li><li> Koru Rag-2 - / - / gc / - otoklavlanmış kafeslerde eksik alıcıları. Hayvan tesisi &quot;temizlik&quot; bağlı olarak, transplantasyon sonrası hayvanların ölümcül radyasyon izleyen 5 hafta boyunca asitlendirilmiş su veya Trimethiprim Sulfasoxazole (Septra) verilmesi gerekebilir. </li><li> Transplantasyon sonrası 3 hafta periferik kan&gt;% 90, GFP + ES-türetilen lökosit bekliyoruz. Lenfositler retroviral HoxB4 IRES-GFP susturmak için Aşı bilinmesine rağmen, çok soyundan olmalıdır. </li></ol>

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>C57Bl/6 Rag-2/gamma-c deficient mice</td>
<td>Animal</td>
<td>Taconic Farm</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Plate shaker</td>
<td>Tool</td>
<td>VWR</td>
<td>33998-360</td>
<td>Orbital Shaker by Ikaworks</td>
</tr>
<tr>
<td>Multi-channel pipettor</td>
<td>Tool</td>
<td>VWR</td>
<td>15000-174</td>
<td>ePet by BioHit</td>
</tr>
<tr>
<td>ES trypsin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>15090-046</td>
<td>0.25% Trypsin in PBS</td>
</tr>
<tr>
<td>Standard trypsin</td>
<td>Reagent</td>
<td>Invtrogen</td>
<td>25300-062</td>
<td>0.05% Trypsin-EDTA</td>
</tr>
<tr>
<td>PBS</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>20012-027</td>
<td>&nbsp;</td>
<tr>
<td>Enzyme-free dissociation buffer</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>13151-014</td>
<td>&nbsp;</td>
<tr>
<td>Dissociation enzyme mix</td>
<td>Buffer</td>
<td>Invitrogen</td>
<td>17104-019</td>
<td>500 mg Collagenase IV</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H2126</td>
<td>freeze in 500 uL aliquots and avoid repeated freezing and thawing.</td>
</tr>
<tr>
<td>DNAse</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D4527</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10-017CV</td>
<td>&nbsp;</td>
<tr>
<td>Protamine sulfate</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P3369</td>
<td>8 ug/mL</td>
</tr>
<tr>
<td>EB differentiation media</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>10% IMDM+cytokines</td>
<td>medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>
<tr>
<td>20% anti-MEM</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Please view recipe on attached Protocol.doc</td>
</tr>		</tbody>
		</table>
		</div>

derivation of hematopoietic stem cells from murine embryonic stem cells

Bir kök hücre kapasitesine sahip bir hücre olarak tanımlanan, hem kendini yenilemek ve birden fazla farklılaşmış döl oluşturmak. Embriyonik kök hücreler (ESC), erken embriyo blastosist türetilmiştir ve pluripotent differentiative yeteneği vardır. Onların engin differentiative potansiyel klinik olarak yararlı bir hücre türleri oluşturmak için onları nasıl ikna edebilmeyi çözmekten merkezli birçok araştırmanın odak yaptı. Fare ESC hematopoietik kök hücrelerin (HSC) başarılı türetme tamamlanmıştır ve bu video protokolde görüntülendi olabilir. HSC, tartışmasız en çok klinik istismar hücre popülasyonu, hematopoetik maligniteler ve bozukluklar sayısız tedavi etmek için kullanılır. Bununla birlikte, birçok hastada HSC tedaviden yarar olabilir uygun donör erişim yoksundur. ESC, bu hastalar için HSC alternatif bir kaynak sağlayabilir. Aşağıdaki protokol ESC-HSC okudu ve insan ESC HSC izole etmek için çabalar bilgilendirmek edilebileceği bir temel kurar. Bu protokol, ESC, piyasada bulunan serum ön elemeden optimal hematopoetik farklılaşma 6 gün embriyoid organları (EBS) olarak ayırt edilirler. EBS sonra ayrışmış ve retroviral HoxB4 ile enfekte. Fare ve ortak kültür varlığı on gün sitokinler teşvik hematopoez - Enfekte EB-elde edilen hücreler, M-CSF / calvaria türetilmiş bir kemik iliği stromal hücre hattı OP9 stroma kaplanmıştır. Bu ko-kültür sırasında, enfekte olan hücreleri nesil 100&#39;ün üzerinde milyonlarca hücreden heterojen bir havuz, büyük ölçüde genişletmek. Bu hücreler daha sonra kurtarma ve sulandırmak ölümcül ışınlanmış fareler için kullanılabilir.

Watch this Scientific Journal Video about Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells at JoVE.com

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Bu protokol detayları fare embriyonik kök hücreler (ESC) ve ölümcül ışınlanmış alıcı farelere sonraki enjeksiyon transplante hematopoietik kök hücre türetme. Kısaca ESC embriyoid organları, sonra retroviral HoxB4 ve OP9 stromal hücreler ve hematopoetik sitokinlerin birlikte kültürlü ile enfekte olarak ayırt edilirler.

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-hematopoietic-stem-cells-from-murine-embryonic-stem

Research

JoVE Journal

Biology

13.2K Görüntüleme.  Harvard Medical School. Bu protokol detayları fare embriyonik kök hücreler (ESC) ve ölümcül ışınlanmış alıcı farelere sonraki enjeksiyon transplante hematopoietik kök hücre türetme. Kısaca ESC embriyoid organları, sonra retroviral HoxB4 ve OP9 stromal hücreler ve hematopoetik sitokinlerin birlikte kültürlü ile enfekte olarak ayırt edilirler.

Video: Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

İnsan Embriyonik Kök Hücre (ES) yayılması

Biyoloji

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

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

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 ...

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Plasenta Hematopoetik Kök Hücre İzolasyonu ve Analizi

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

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 ...

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

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


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

Murin Yolk Sac ve AGM Erken Hematopoetik Kök Hücre İzolasyonu

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

Isolation 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 ...

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 ...

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 ...

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

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

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

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

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